A bienzyme fiberoptic sensor for the chemiluminescent flow injection analysis of L-lactate was developed. Lactate oxidase and peroxidase were covalently immobilized on preactivated polyamide membrane either separately on different membranes or randomly coimmobilized on the same membrane. Hydrogen peroxide, generated by the lactate oxidase reaction in the presence of L-lactate, was the substrate of the second reaction catalyzed peroxidase, in the course of which light was emitted in the presence of luminol. Compartmentalization of the enzyme layer was obtained by stacking a peroxidase membrane on a lactate oxidase membrane at the sensing tip of the fiberoptic sensor. When using such a sensing tip, the response of the fiberoptic sensor for lactate was 22 times higher than the response obtained when using a membrane bearing the two enzymes randomly coimmobilized. With the compartmentalized sensor tip, the detection limit was 250 pmol lactate and the coefficient of variation was 1.7% for 10 replicates of 6.25 nmol lactate. The method was applied for lactate determination in reconstituted whey solutions, and the results were in good agreement with the analysis by an enzyme electrode.