© 2021, King Abdulaziz City for Science and Technology.New three-enzyme lactose amperometric biosensor based on platinum disc electrode modified by nanosized semipermeable poly (meta-phenylenediamine) film was developed in this work. It was studied the influence of different conditions of meta-phenylenediamine electropolymerization on the parameters (sensitivity and selectivity) of the amperometric transducers. The most optimal parameters were achieved after performing the electropolymerization of 5 mM meta-phenylenediamine applying 10–13 cyclic voltammograms. The bioselective element of lactose biosensor consisted of three enzymes (β-galactosidase, mutarotase, glucose oxidase) that were immobilized on the surface of modified working electrode. The analytical characteristics of the developed biosensor were studied. The linear range of lactose determination extended from 0.01 mM to 1.25 mM. The response time of the biosensor was 30 s. The biosensor showed high signal reproducibility (RSD = 1.16%), high sensitivity (LOD = 0.005 mM) and high selectivity. The proposed biosensor was tested for measurement of lactose concentration in milk samples. The high correlation between results obtained with lactose biosensor and HPLC was shown (R = 0.96%). Thus, the developed biosensor is suited for rapid, inexpensive, sensitive, selective and simple determination of lactose in milk samples.