The electrochemical properties of a Prussian blue (PB) electrode were improved by introducing cetyltrimethylammonium bromide (CTAB) and Au nanoparticles (AuNPs) into PB films. The novel hybrid films (PB/CTAB/AuNPs) were fabricated by electrodepositing PB and AuNPs in the presence of CTAB. The electrochemical behavior of the hybrid film in some supporting electrolyte (cations for the K+, Na+, or K+/Na+) was investigated in detail, and well-defined and reversible voltammetric responses were obtained in Na+-based electrolytes. The catalytic activity of the PB/CTAB/AuNPs electrode toward hydrogen peroxide (H2O2) reduction at a neutral pH was also investigated, and the results indicated that the electrochemical reduction of H2O2 in the presence of physiological levels of Na+ was superior to that of a PB-modified electrode. Moreover, the PB/CTAB/AuNPs electrode exhibited good performance, a low detection limit (0.1 mu M), and high stability at a wide range of concentrations (0.882-195 mu M). To determine the performance of PB nanocomposite electrodes in Na+-based phosphate buffers, an amperometric biosensor with a PB/CTAB/AuNPs electrocatalyst was developed. To fabricate this sensor, the enzyme was immobilized in sol-gel and was electrodeposited onto a PB nanocomposite film. The results indicated that the biosensor can be used at a wide range of concentrations (20-400 mu M) and possesses a low detection limit (7 mu M) for glucose. These characteristics demonstrate that PB nanocomposite film can be used as an electron mediator for biosensors in potassium-free phosphate buffers.