Abstract: In a traditional epi-illumination fluorescence microscope, the detection path is coaxial with the illumination path, which induces the non-focal plane fluorescence and deteriorates the imaging quality. Light sheet fluorescence microscopy (LSFM), differing from the traditional fluorescence microscopes, adopts an orthogonal configuration of detection and illumination paths. A thin sheet is formed from the excitation beam, which only excites a single layer of the sample. This methodology prevents the excited fluorescence from the non-focal plane during imaging. Besides, the utilization of the laminar illumination light can significantly reduce the exposure time of the fluorescence imaging. As a result, the effects of photobleaching and phototoxicity are decreased. In this review, we first introduce the basic light path structure compositions of a LSFM system as well as the optimization and innovation based on these structures. Next, we discuss enormous processing methods developed for samples both in vitro and in vivo. Benefiting from all these innovations, LSFM outstands in performing the 3D imaging of the fluorescence-labeled biological samples and can function steadily for a long recording time. Finally, we propose potential researching directions in the future, and discuss the technical limitations of current LSFM. This review aims to provide researchers in the relevant scientific research fields with a comprehensive understanding and inspiring reference of LSFM techniques.