A new L-phenylalanine dehydrogenase (PHD) and Toluidine Blue O (TBO) based amperometric biosensor for the determination of L-phenylalanine was reported in this study. An amperometric L-phenylalanine biosensor with immobilization of TBO (as a mediator,), PDH and nicotinamide adenine dinucleotide (NAD(+)) onto polypyrrole-polyvinylsulphonate (PPy-PVS) film was accomplished on the surface of a platinum electrode. Cross-linking with glutaraldehyde technique was used for immobilizing PDH on PPy-PVS film. Toluidine Blue O (TBO) was used as the mediator. The changes in the anodic current at -0.195 V vs Ag/AgCl produced by TBO was proportional to the L-phenylalanine concentration changes in the sample. The effects of substrate concentration, pH and temperature on the response of the L-phenylalanine biosensor were investigated. Optimum pH and temperature were found to be 7.0 and 60 degrees C, respectively. The linear working region of biosensor is 5.0 x 10(-7) - 2.5 x 10(-5) M (R-2 = 0.9816). The detection limit of the biosensor was 1.0 x 10(-8) M and response time of the biosensor was 200 s. The storage stability and application stability of the biosensor were also studied. Several possible interfering substances' effects on the phenylalanine biosensor were investigated. The developed biosensor was tested in determinations of content in a synthetic blood sample.