Gradient strengthening layer will emerge on the grinding surface of Inconel 718 due to the difference of microstructure. The surface microstructure and microhardness are not independent of each other, and the microhardness is the embodiment of the microstructure evolution in the strength aspect. In this paper, the microstructure observation, microhardness experiments and strengthening theory were combined to analyze. The experimental results show that the grinding surface consists of grain refinement layer and high-density dislocation layer. The grain refinement layer is constituted of equiaxed nano-grains and elongated grains, in which grain boundary strengthening occurred leading to an increase in microhardness. Dislocation strengthening occurred in the high-density dislocation layer, in which the increment of dislocation density is approximately 3.54 × 109 mm− 2 compared with inner matrix. Microhardness of high-density dislocation region reaches the maximum (438.6 ± 11.1 HV0.01) because of the dislocation strengthening. The variation of microhardness is discussed from two strengthening mechanisms of grain boundary strengthening and dislocation strengthening, and the strengthening mechanism in the different regions of grinding surface is revealed. The calculated microhardness increments through these mechanisms in the refined-grain region and the high-density dislocation region are basically consistent with the measured values.