As an important branch of nonlinear optics, second harmonic generation (SHG) is becoming one of the most important means to characterize crystal structure. Among various methods of characterization, because of nondestructive detection, high stability, tunability, ultrafast response, polarization sensitivity, versatility and simplicity, SHG is widely used to characterize the structure of two-dimensional (2D) materials. It provides important information for the physical properties and functional applications of 2D materials, as well as greatly promotes the rapid development of basic research on 2D materials. Here, the current state of the art focuses on the recent research work of SHG in 2D material structure characterization. Firstly, the principle of the second harmonic generation is briefly introduced. Then, the second harmonic generation device with femtosecond laser connected to confocal Raman spectrometer is taken as an example to present the mechanism of SHG. Afterwards, the applications of SHG are demonstrated in the thickness of interlayer stacking of 2D materials, the stacking angle between different layers of 2D materials, the grain boundary and the crystal orientation of monolayer 2D materials. The second harmonic intensity is used as a direct and sensitive means to monitor the strain amplitude, and the SHG signal changes are used to track defects in materials. Meanwhile, the importance of multi-dimensional correlation analysis of second harmonic generation, Raman spectroscopy and photoluminescence in comprehensive and in-depth characterization of materials is also explored. Finally, the potential research directions and prospects based on SHG in material characterization in the future is prospected.