The design of the bioactive sensing laver plays a major role in the performance of a biosensor especially when it is based on a multienzymatic sequential reaction. Generally, the different enzymes are coimmobilized on the same support but a new approach consisting of tile compartmentalization of the sensing layer enables improvement in the performance of the biosensor. This approach has been applied to the development of a trienzymatic sensor for the bioluminescent microassay of D-sorbitol. The sequential reaction involves sorbitol dehydrogenase, NAD(P)H:FMN oxidoreductase, and bacterial luciferase. The hyperconcentration effect of the common intermediate, NADH, resulting from the compartmentalization of the enzymatic layer has been experimentally evidenced. The selection of a well-balanced ratio of the different enzymes in the compartmentalized layer is essential to strongly improve the biosensor sensitivity. A 14.5-fold enhancement of the biosensor sensitivity has been obtained compared to the value observed with a coimmobilized system. The dynamic linear range of measurements extends from 50 nM to 2 mu M with a response time of 4-6 min. Both the reproducibility of the measurements and the stability of the enzymes are satisfactory enough to make the biosensor operational for use with real samples.