The cartoon was produced entirely in the United States (the only series that does this full time) using a combination of Macromedia Flash and traditional cel animation. Each program was 30 minutes long (with commercials) and featured three seven-minute segments that borrow inspiration from Japanese animation.
During the first season, the show included short clips of the real Ami and Yumi making childish commentary (in English and non-subtitled Japanese) at the beginning and end of each episode. They only performed short clips at the beginning of the show during the second and third seasons. Starting with the second season, the duo was sometimes shown holding title cards introducing the cartoon segments. At the end of the episode "Sitcomi Yumi", Ami and Yumi watch TV and see the animated Kaz with the real Ami and Yumi.
The real Puffy AmiYumi performs the cartoon's theme song (which is also in Japanese, German, Spanish, and Portuguese in the respective regions), and many episodes feature one or more of the duo's songs playing in the background, along with music by their Puerto Rico muse Andy Sturmer.
The animated Puffy AmiYumi travel all over the world in their tour bus. While appearing the same size as a regular bus on the outside, it appears to have enough internal space to house the girls' rooms (including full-sized beds), Kaz' room, their equipment, and several televisions and computers, among other things. In the episode "Domo", Kaz refers to an upstairs area. It also seems capable of running on autopilot, as many episodes have Kaz, Ami, and Yumi sitting in the rear cabin of the bus while traveling. Several times, the rear door has been opened to receive packages, which a boy on a scooter usually delivers.
Though the characters speak English, the script occasionally intersperses their vernacular with Japanese speech, especially when the characters react to events that they find to be surprising. Calling out "Tasukete!" instead of "Help!" is commonly used. During production of the series, DeLisle learned some Japanese from Kawaye and Young, both of whom speak the language fluently.