We present a light sheet fluorescence microscopy (LSFM) with active optical manipulation by using linear optical tweezers (LOTs). In this method, two coaxially transmitting laser beams of different wavelengths are shaped using cylindrical lenses to form a linear optical trapping perpendicular to the optical axis and an excitation light sheet (LS) parallel to the optical axis, respectively. Multiple large-sized polystyrene fluorescent microspheres are stably captured by LOTs, and their rotation angles around specific rotation axes are precisely controlled. During a sample rotation, the stationary excitation LS scans the sample to obtain fluorescence sectioning images of the sample at different angles.