The autofocus method has been investigated to improve the precise positioning of a substrate surface at the center of the laser focal spot in two-photon polymerization (TPP) nanofabrication. For this purpose, we developed a novel autofocus method using normalized image size, which was calculated with the second momentum radius (SMR) of two-photon induced fluorescence (TPIF). The SMR of TPIF was theoretically analyzed and experimentally compared with the average intensity of TPIF for various input laser powers. The results show that the proposed method enhanced the precision and robustness of autofocus in TPP. Specifically, the experimental creation of ascending voxel arrays demonstrated both the method's immunity to input laser power change, and a high precision of +/- 0.045 mu m. To test the practical feasibility of the proposed autofocus method, 300 mu m x 260 mu m single-layer honey-comb structures were successfully fabricated with precompensation and dynamic compensation using the proposed autofocus method.