Functional carbon nanomaterials are significantly important for the development of high performance sensitive and selective electrochemical biosensors. In this study, graphene supported platinum nanoparticles (GN–PtNPs) and nitrogen doped graphene supported platinum nanoparticles (N-GN– PtNPs) were synthesized by a simple chemical reduction method and explored as high performance nanocatalyst supports, as well as doped nanocatalyst supports, toward electrochemical oxidation of homocysteine (HCY) for the first the time. Our studies demonstrate that N-doped graphene supported PtNPs show higher electrocatalytic activity for HCY with an experimental detection limit of 200 pM. Moreover, N-doped graphene supported Pt was demonstrated to have excellent selectivity in the electrochemical oxidation of HCY i.e., the detection of HCY is successful in the presence of a 20-fold excess of ascorbic acid (AA). The practical application of N-doped graphene supported PtNP materials is effectively shown for the determination of HCY in both human blood serum and urine samples, by differential pulse voltammetry under optimized conditions. Our findings conclude that N-doped graphene supported PtNPs can be developed as a high performance and versatile nano-electrocatalyst for electrochemical biosensor applications.