In the present work, we report the selective and sensitive electrochemical detection of L-dopa using crude Polyphenol oxidase enzyme (PPO) immobilized on electrochemically reduced graphene oxide–silver nanoparticles (RGO-Ag) nano composite modified graphite (Gr) electrode. The Gr electrode is first modified through the electrochemical reduction of graphene oxide - silver (GO-Ag) nano composite to RGO-Ag nano composite on graphite electrode. The crude extract of PPO from Manilkara Zapota (sapota) fruit is later immobilized onto Gr/RGO-Ag modified electrode. Modification of Gr electrode at each step is confirmed by cyclic voltammetry (CV) and Electrochemical impedance (EIS) techniques. The surface characterization of modified electrode is performed by Scanning electron microscopy (SEM). The developed sensor exhibited excellent electrocatalytic activity towards the detection of L-dopa in the concentration range of 1–150 μM with low detection limit and high sensitivity. Further, the practical applicability of the developed biosensor is examined for the detection of L-dopa in urine samples.