In this paper, a novel amperometric L-glutamate (Glu) biosensor with immobilization of L-glutamate oxidase (L-GlOx) on polypyrrole-polyvinylsulphonate (PPy-PVS) film has been successfully developed. L-GlOx enzyme was immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA) and bovine serum albumin (BSA). Determination of Glu was carried out by oxidation of enzymatically produced H2O2 at 0.3 V versus Ag/AgCl. The optimum pH and temperature parameters were found to be 9.0 and 55 degrees C, respectively. There were three linear parts in the regions between 1.0 x 10(- 9) and 1.0 x 10(- 8) M (R-2 = 0.847), 5.0 x 10(- 8) and 5.0 x 10(- 7) M (R-2 = 0.997), 5.0 x 10(- 7) and 5.0 x 10(- 5) M (R-2 = 0.994). Storage stability, operation stability of the enzyme electrode were also studied.