The effect of laser peening on vibration fatigue performance of electrochemically hydrogenated 316L austenitic stainless steel was studied. The residual stress, microhardness and microstructure of laser peened specimens with different power densities followed by hydrogen charging were tested and analyzed, and the vibration fatigue life and fracture morphology of the corresponding specimens were compared and studied. The results show that laser peening induces an increase in dislocation density on the surface of material and effectively refine the grains, which inhibits the invasion of the hydrogen atom. At the same time, the complex grain boundary and high density dislocation multiplication structure hinder the aggregation and diffusion of hydrogen atoms, reduce the degree of the martensitic transformation of 316L austenitic stainless steel, which helps to suppress micro-crack initiation. On the other hand, the high residual compressive stress induced by laser peening not only inhibits hydrogen penetration, but also increases the fatigue crack growth threshold and slows down the crack growth rate. Vibration fatigue test results show that the fatigue life of laser peened specimens with different power densities followed by hydrogen charging has been significantly improved, and the maximum amplitude can be up to 79.36%. The fracture morphology analysis further proves that laser peening can effectively reduce the fatigue crack growth rate of hydrogen-charged specimens, increase the fracture toughness of the material, and then improve the vibration fatigue properties of materials.