It is of great significance for structural design and engineering evaluation to obtain the elastic–plastic parameters of materials. The inverse estimation of elastic–plastic parameters of materials based on nanoindentation technology has been applied in many pieces of research, but it has proved to be difficult to determine the elastic–plastic properties of materials by only using a single indentation curve. A new optimal inversion strategy based on a spherical indentation curve was proposed to obtain the elastoplastic parameters (the Young’s modulusE, yield strengthσy, and hardening exponentn) of materials in this study. A high-precision finite element model of indentation with a spherical indenter (radius R = 20 µm) was established, and the relationship between the three parameters and indentation response was analyzed using the design of experiment (DOE) method. The well-posed problem of inverse estimation under different maximum indentation depths (hmax1= 0.06 R,hmax2= 0.1 R,hmax3= 0.2 R,hmax4= 0.3 R) was explored based on numerical simulations. The results show that the unique solution with high accuracy can be obtained under different maximum press-in depths (the minimum error was within 0.2% and the maximum error was up to 1.5%). Next, the load-depth curves of Q355 were obtained by a cyclic loading nanoindentation experiment, and the elastic–plastic parameters of Q355 were determined by the proposed inverse-estimation strategy based on the average indentation load-depth curve. The results showed that the optimized load-depth curve was in good agreement with the experimental curve, and the optimized stress–strain curve was slightly different from the tensile test, and the obtained parameters were basically consistent with the existing research.