Light sheet microscope is an important tool for three-dimensional imaging owing to its powerful optical section capability, fast imaging speed, and low phototoxicity. This microscope typically uses two objectives that are placed perpendicular to each other for illumination and imaging; however, this structure imposes spatial constraints and prohibits the use of an imaging objective with high numerical aperture. These limitations were resolved using single-objective light sheet microscopy techniques, such as oblique plane illumination and micromirror microdevice reflection, demonstrating potential for high-resolution and volumetrically high-speed imaging. Furthermore, these techniques can be combined with various other techniques, such as super-resolution microscopy, which has made great progress in recent years. This review article introduces the principles, key performance enhancements, and biomedical applications of single-objective light sheet microscopy.