A biosensor using permeabilized yeasts (Hansenula anomala) is described for the determination of L-lactate. The same electrode can be used either potentiometrically or amperometrically. The linear ranges are respectively of 4.10−5M to 2.10−3M for potentiometry and 8.10−7M to 3.10−3M for amperometry with a reproducibility and a precision of ± 2 to 3%. The parameters involved in the optimization of electrode response, such as pH, temperature, cofactor concentration and ionic strength of the buffers, are discussed in details for both types of measurements. The amperometric technique is suitable for lactate determinations in biological media. In this case, differential measurements are used in order to eliminate interferences of biological redox reactions.