Nonlinear optics, which is a subject for studying the interaction between intense light and materials, has great impact on various research fields. Since many structures in biological tissues exhibit strong nonlinear optical effects, nonlinear op-tics has been widely applied in biomedical studies. Especially in the aspect of bio-imaging, nonlinear optical techniques can provide rapid, label-free and chemically specific imaging of biological samples, which enable the investigation of bio-logical processes and analysis of samples beyond other microscopy techniques. In this review, we focus on the introduc-tion of nonlinear optical processes and their applications in bio-imaging as well as the recent advances in this filed. Our perspective of this field is also presented.