Second harmonic generation (SHG) is a novel optical imaging technology that has been recently developed. SHG has attracted considerable attention as a new tool for the biological structure and durable tracking. The SHG technology eliminates many disadvantages associated with the classical fluorescent probes. It is an ideal in vivo imaging method and exhibits good biomedical application prospects. In this study, we introduce the principle of SHG and its imaging device, classify the SHG media, review the application of SHG in biomedical imaging, and prospect future opportunities and challenges.