The embodiment of the invention discloses a super-resolution microscopic imaging system. The system comprises an excitation light source generation module, an imaging module, a focal plane locking module and a control and data acquisition module, wherein the excitation light source generation module is used for generating an excitation light source; the imaging module is used for receiving the excitation light source, enabling the excitation light source to irradiate a sample on the sample slide to enable a view field area of the sample to generate fluorescent light, and collecting the reflected fluorescent light through the camera; the focal plane locking module is used for emitting laser to the imaging module and receiving the laser reflected by the imaging module so as to lock the distance between an objective lens in the imaging module and a sample slide; and the control and data acquisition module is used for controlling and adjusting optical elements in the excitation light source generation module, the imaging module and the focal plane locking module, and acquiring fluorescence data obtained by the imaging module for data analysis. According to the system, a compact, adjustable and controllable single-molecule positioning microtechnique with high automation degree is realized, and the precision and the accuracy are improved.