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 optics 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 biological processes and analysis of samples beyond other microscopy techniques. In this review, we focus on the introduction 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.