In this work, a novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide (RGO-AuNPs) and l-lactate dehydrogenase (LDH) was developed for the sensing of l-lactate. Firstly, the RGO-AuNPs modified screen printed electrodes were tested for NADH detection showing a wide dynamic range and a low detection limit. Next, the biosensor was constructed by incorporating both enzyme and RGO-AuNPs in a sol gel matrix derived from tetrametoxysilane and methyltrimetoxysilane. The enzyme loading, working pH, and coenzyme concentration were optimized. The biosensor linearly responded to l-lactate in the range of 10µM-5mM and showed a good specific sensitivity of 154µA/mMcm(2) with a detection limit of 0.13µM. This was accompanied by good reproducibility and operational stability. Tests on artificial serum proved that l-lactate can be determined practically without interferences from commonly interfering compounds such as urate, paracetamol and l-ascorbate. Our LDH/RGO-AuNPs/SPCE based biosensor thus performs as electrochemical device for the detection of l-lactate as a viable early cancer bio-marker.