A surface plasmon resonance (SPR) based biosensor for glucose is presented in which a thin gold film modified with polypyrrole and glucose oxidase (PPy-GOx) acts as the sensor chip. It is based on SPR response to the change of refractive index of PPy-GOx film by the enzymatic catalytic reaction. The co-electropolymerization of pyrrole and GOx was carried out under cyclic voltammetric conditions, and simultaneously monitored by in-situ SPR. It has been revealed that the enzymatic reaction between GOx and PPy in the presence of glucose can lead to distinct changes in the SPR signal. From the experiments, a linear relationship was obtained in the range 1–100 μmol L−1 between glucose concentration and the rate of redox transformation of PPy. The detection limit was 0.5 μmol L−1 (S/N = 3) and recoveries were 95.2–102.7%.