Herein, development of an enzymatic biosensor for rapid quantitation of the disaccharide lactose in dairy samples (whey permeates and milk protein isolates (MPI)) is presented. Biosensor fabrication involved a chitosan/enzyme/crosslinker configuration with enzymes glucose oxidase and β-galactosidase at platinum and glassy carbon electrodes. Solution phase mediation was used to lower the operating potential (Eapp = 0.3 V vs Ag/AgCl), minimising any contribution from endogenous background electroactive species. Potential sweep and potentiostatic experiments realised analytical data for the lactose sensor with linear range 5.83 × 10−3 to 1.65 × 10−2 M, sensitivity 9.41 × 10−4 C cm−2 mM−1 and LOD of 1.38 mM. Scanning electrochemical microscopy realised surface characterisation of the enzyme layers with approach curves and redox competition mode imaging achieved over the active enzyme bilayer. Both glucose and lactose sensing was realised in whey permeate sample measuring 23.7 mM lactose, correcting for free glucose contributing signals, with 92.2% correlation with results obtained from the sample certificate of analysis. Solution phase mediation at glassy carbon enzyme electrodes resulted in lactose quantitation in milk protein isolates, measuring 1.16 mM for the MPI 1 (low lactose) and 1.54 mM for the MPI 10 (standard) sample with validation by HPLC analysis.