Lactose biosensor based on surfactant doped polypyrrole (PPy) was developed. Galactose oxidase and β-galactosidase was coimmobilized in PPy matrix during electropolymerization process with the presence of sodium dodecylbenzene sulphonic acid as surfactant. Bi-enzyme entrapped PPy was characterized with Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV), and scanning electron microscopy (SEM). The response of the enzyme electrode was measured by CV in the range of −0.1 to 1.0 V versus Ag/AgCl which was due to the electrooxidation of enzymatically produced H2O2. The effect of lactose concentration was investigated. Response time of biosensor was found to be 8–10 s (the time required to obtain the maximum peak current) and upper limit of the linear working portions was found to be 1.22 mM lactose concentration with a detection limit of (2.6 × 10−6 M). The apparent Michaelis–Menten constant was found to be 0.117 mM lactose. The effects of interferents (ascorbic acid and uric acid) were determined. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40200.