L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2(Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeastOgataea polymorpha.The possibility of direct electron transfer from the reduced form of Fcb2to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M−1·m−2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2and the hexacyanoferrate of gold, having a sensitivity of 253 A·M−1·m−2without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.