A bioluminescent fiber optic sensor for the analysis of L-lactate is described. It is based on a reaction sequence catalyzed by three different enzymes: luciferase fromV. harveyi, NAD(P) H: FMN oxidoreductase fromV. fischeriand lactate dehydrogenase from rabbit muscle covalently immobilized on polyamide membranes. Two kinds of sensing layer were studied, one consisting of only one membrane on which the enzymes were randomly coimmobilized, the other being a compartmentalized system obtained by stacking a luciferase/oxidoreductase membrane on a lactate dehydrogenase membrane. After optimization, the performances of the biosensor in terms of sensitivity, detection limit and dynamic linear range of measurements were strongly improved by compartmentalization of the sensing layer compared with those obtained using the coimmobilized system. A fivefold increase in biosensor sensitivity was obtained and the detection limit was 0.2 μM lactate.