Two-photon microscopy’s ability to maintain good spatial resolution in thick biological tissues has led to its application in in-vivo brain imaging studies soon after its conception. As neural networks have cross-scale multidimensional spatio-temporal properties, two-photon microscopy has developed rapidly and significantly in recent years to meet the demand for in-vivo cross-scale imaging of the brain. This paper firstly introduces the working principle of two-photon microscopy, then reviews the progress of two-photon microscopy from five perspectives: imaging field of view, imaging flux, imaging depth, resolution, miniaturization, and analyzes the difficulties and future challenges of cross-scale two-photon in-vivo microscopic imaging technology.