A two-photon stimulated emission depletion composite microscope, comprising a two-photon imaging unit (100) and an STED imaging unit (200), wherein the two-photon imaging unit (100) can be used for a relatively thick sample, and the STED super-resolution imaging unit can be used for a region of interest on a surface of a sample, and the microscope makes light spots generated by an excitation light and a depletion light after being focused by an objective lens (OL) accurately coincide in a three-dimensional distribution. The two-photon stimulated emission depletion composite microscope (10) integrates two functions of STED imaging and two-photon imaging and makes the two types of light spots generated by an excitation light and a depletion light after being focused by an objective lens accurately coincide in a three-dimensional distribution, thereby providing a powerful tool for cutting-edge biomedical research.