Organisms are composed of molecules typically arranged and oriented in order. The structures of organisms are related to the positional distribution of molecules and their spatial orientation. Polarized fluorescence microscopy (PFM) is such an imaging tool that facilitates the sense and observation of the molecule orientation in biological samples by utilizing fluorescence polarization, enabling the revealing of functional and metabolic information beyond fluorescence intensity provided by more traditional fluorescence microscopy. In this review, we first briefly describe the theory and principle of PFM and then introduce several implementations of PFM on different microscopic modalities. We later summarize its applications in the field of biomedical research and finally discuss its potential future directions.