A L-lactate oxidase (LOD)-based biosensor is developed for the determination of L-lactate in blood samples. The L- lactate oxidase membrane is prepared by covalently linking LOD into a nylon set, followed by attaching the membrane onto a flow injection type of oxygen electrode. The response of the biosensor is based on the limited diffusion of L- lactate on the L-lactate oxidase membrane. No performance difference have been found between the LOD-based biosensor and regular enzyme optical determination methods for blood sample testing. It is suggested that the LOD-based biosensor may serve as an alternative for the detection of L-lactate in blood.