An amperometric enzyme electrode for L-Alanine determination was developed. The probe is based on a platinum electrode which senses the hydrogen peroxide produced by the enzymatic reactions catalyzed by two enzymes: alanine aminotransferase (ALT) and glutamate oxidase. Both the enzymes were immobilized on polymeric membrane supports. Interferences were discriminated by additional protective membranes. Buffer, pH, cosubstrate, temperature and immobilization procedures were optimized. The probe response was linear in the concentration range 10−5– 10−3mol/L with a detection limit of 2·10−6mol/L.