The series revolves around Dexter (voiced by Christine Cavanaugh in seasons 1–2; Candi Milo in seasons 3–4), a bespectacled boy-genius who possesses a secret laboratory hidden behind a bookcase in his bedroom. The laboratory is filled with Dexter's inventions and can be accessed by speaking various passwords or by activating hidden switches on Dexter's bookshelf (e.g. pulling out a specific book). Though highly intelligent, Dexter often fails at what he has set out to do when he becomes overexcited and makes careless choices. Although he comes from a typical all-American family, Dexter speaks with a thick accent of indeterminate origin. Christine Cavanaugh described it as "an affectation, some kind of accent, we're not quite sure. A small Peter Lorre, but not. Perhaps he's Latino, perhaps he's French. He's a scientist; he knows he needs some kind of accent." Genndy Tartakovsky explained, "He considers himself a very serious scientist, and all well-known scientists have accents."

Dexter manages to keep the lab a secret from his clueless, cheerful Mom (voiced by Kath Soucie) and Dad (voiced by Jeff Bennett), who never take notice of it. However, he is frequently in conflict with his hyperactive, annoying older sister, Dee Dee (voiced by Allison Moore in seasons 1 and 3; Kat Cressida in seasons 2 and 4). In spite of Dexter's advanced technology, Dee Dee eludes all manner of security, and once inside her brother's laboratory, she delights in playing haphazardly, often wreaking havoc with his inventions. Though seemingly dim-witted, Dee Dee often outsmarts her brother and even gives him helpful advice. For his part, Dexter, though annoyed by his intrusive sibling, feels a reluctant affection for her and will come to her defense if she is imperiled.

Dexter's nemesis is a rival boy-genius from his school named Susan "Mandark" Astronomonov (voiced by Eddie Deezen). Just like Dexter, Mandark also has his own laboratory, but his schemes are generally evil and designed to gain power while downplaying or destroying Dexter's accomplishments. In the revival seasons, Mandark becomes significantly more evil, becoming Dexter's enemy rather than his rival and his laboratory changing from brightly-lit with rounded features to gothic-looking, industrial, and angular. Because Dexter's inventions are often better than his, Mandark tries to make up for this by stealing Dexter's plans. Mandark's weakness is his love for Dee Dee, though she ignores him and never returns his affections.

Recurring segments
Almost every episode of Dexter's Laboratory is divided into three different stories/segments, each one being approximately 8 minutes in length. Occasionally, the middle segment centered on characters from the Dexter's Laboratory universe other than Dexter and his family. Two of these segments were shown primarily during the first season: Dial M for Monkey and The Justice Friends. Dial M for Monkey was the middle segment for the first six episodes of season one, and The Justice Friends took its place for the rest of the season.

Dial M for Monkey
The Dial M for Monkey shorts feature Dexter's pet laboratory monkey named Monkey (vocal effects provided by Frank Welker), whom Dexter believes is an ordinary monkey and nothing more. However, Monkey secretly has superpowers and fights evil as the superhero Monkey. Monkey is joined by his partner Agent Honeydew (voiced by Kath Soucie) of Global Security, her director the Commander General (voiced by Robert Ridgely in Season 1, Earl Boen in Season 2), and a team of assembled superheroes. Dial M for Monkey was created by Genndy Tartakovsky, Craig McCracken, and Paul Rudish.

The Justice Friends
The Justice Friends consists of Major Glory (voiced by Rob Paulsen), Valhallen (voiced by Tom Kenny), and the Infraggable Krunk (voiced by Frank Welker), three superheroes who are all roommates living in an apartment complex called Muscular Arms. Most of the trio's adventures deal less with their lives as superheroes and more with their inability to get along as roommates; it is presented as a sitcom, including a laugh track. Genndy Tartakovsky's inspiration for The Justice Friends came from reading Marvel Comics when he was learning how to speak English. Tartakovsky stated in an interview with IGN that he was somewhat disappointed with how The Justice Friends turned out, saying, "it could have been funnier and the characters could have been fleshed out more."
Mini-segments
Between the three main segments in seasons one and two are brief mini-segments, many of which feature only Dexter and Dee Dee. Other characters from the series may star in them also, such as "The Puppet Pals", two live-action puppets named Puppet Pal Mitch (Rob Paulsen) and Puppet Pal Clem (Tom Kenny).

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