L-proline assisted silver nanoparticles were electrodeposited on glassy carbon electrode by one-step voltammetry method. The results of Raman spectroscopy, X-ray photoelectron spectroscopy and high-resolution transmission electron microscopy confirmed a uniform distribution of nanometer sized silver nanoparticles in the presence of L-proline and the formation of mixed silver oxides in the absence. L-proline as a stabilizer plays the very important role in slowing the oxidation of metallic silver and makes contributions to avoid aggregations. More importantly, L-proline caused nearly 200 mV potential shifted towards less negative potential and almost twofold sensitivity increased. This low-cost and simple silver nanoparticles sensor is not only free from various disadvantages of enzyme or noble based sensors, but also provides a superior sensing performance for electrochemical detection of hydrogen peroxide. The resultant silver nanoparticles stabilized using L-proline displayed lower detection limit of 0.05 mu M, larger detection linear range from 0.1 mu M to 5145 mu M, and higher selectivity in the presence of interfering species such as ascorbic acid, uric acid, L-glucose, glycine and lactose, for H2O2 detection, relative to silver nanoparticles without L-proline. These comprehensive characterizations attribute the excellent H2O2 electrocatalytic performance to its fast electron transfer kinetics of the metallic silver (Ag-0) in L-proline stabilized silver nanoparticles. The fabricated sensor also reliably applied in detection of H2O2 in milk samples. This work suggests the L-proline assisted silver nanoparticles are promising materials for highly active and selective non-enzymatic H2O2 detection.