In this work, we report an electrochemical surface plasmon resonance/waveguide (EC-SPR/waveguide) glucose biosensor that could detect enzymatic reactions in a conducting polymer/glucose oxidase (GO(x)) multilayer thin film. In order to achieve a controlled enzyme electrode and waveguide mode, GO(x) (negatively charged) was immobilized with a water-soluble, conducting N-alkylaminated polypyrrole (positively charged) using the layer-by-layer (LbL) electrostatic self-assembly technique. The electrochemical and optical signals were simultaneously obtained from the composite LbL enzyme electrode upon the addition of glucose as mediated by the electroactivity and electrochromic property of the polypyrrole layers. Signal enhancement in EC-SPR detection is obtained by monitoring the doping-dedoping events on the polypyrrole. The real-time optical signal could be distinguished between the change in the dielectric constant of the enzyme layer and other nonenzymatic reaction events such as adsorption of glucose and the change of the refractive index of the solution. This was possible by correlation of both the SPR mode and the m = 0 and 1 modes of the waveguide in an SPR/waveguide spectroscopy experiment.