To improve the surface integrity, the effects of Ultrasonic Surface Rolling Processing (USRP) technology on the micro-gradient structure and mechanical properties of 45 steel is studied. The micro-gradient structure induced by USPR was analyzed by optical microscope (OM), scanning electron microscope (SEM) and electron back scattering diffraction (EBSD). Besides, the change of surface roughness and microhardness after USPR were also investigated. Finally, considering the scale effect, the instrumented indentation method modified by the strain gradient theory was used to analyze the gradient mechanical properties of the treated 45 steel. The results showed that a zone containing fine grains of different orientations was formed in the surface layer of the specimen with the depth of 120 μm. The grain size varies in a gradient, and the average grain size in the region of 10 μm from the surface layer can reach 360 nm. The number of small and large angular grain boundaries in the surface layer of the specimen increased significantly. The surface roughness of the specimen was reduced to the nanometer level, which was 96.7% lower than the original roughness, and the surface microhardness was increased by up to 55.1%. According to the degree of plastic deformation, the surface microstructure was divided into three areas:strongly deformed area, micro-deformed area and unaffected area. The depth of strongly deformed area and micro-deformed area increased with the number of processing passes, and the depth of plastic deformed layer was up to 320 μm. Both the elastic and plastic properties of the surface layer underwent significant gradient changes, with the elastic modulus increasing by up to 1.67 times, the yield strength increasing by up to 83.3% and the work hardening index decreasing by up to 68.3%, which are effective in improving the fatigue and corrosion resistance of the material.