Laser scanning confocal microscope (LSCM) is one of the most important tools for biological imaging due to its strong optical sectioning capability, high signal-to-noise ratio, and high resolution. On the basis of LSCM, line-scanning fluorescence microscopy (LSFM) uses linear scanning instead of point scanning to improve the speed of image acquisition. It has the advantages of simple system structure, fast imaging speed, and weak phototoxicity, and in addition, it is more suitable for high-resolution and fast imaging of living thick samples. It is of great significance for studying the life science, biomedicine, and others. However, the current LSFM technology still faces many urgent problems in terms of system flexibility, imaging speed, resolution and optical sectioning capabilities. Therefore, based on the existing multifocal structured illumination microscopy (MSIM) in our laboratory, a digital line-scanning fluorescence microscopy (DLSFM) based on digital micromirror device(DMD) is presented in this paper. In the illumination path, a high-speed spatial light modulator DMD is adopted to realize multi-line parallel scanning excitation, which simplies the optical system and improves the flexibility and scanning speed of the system. A DLSFM image reconstruction algorithm based on the standard deviation of fluorescence signal is proposed, which is combined withthree-dimensional (3D) Landweber deconvolution algorithm to achieve 3D high-resolution optical slice image reconstruction. On this basis, the imaging experiments on fluorescent beads and standard samples of mouse kidney section are carried out by using DLSFM. The experimental results show that the resolution of DLSFM in the x, yand zdirections is 1.33 times, 1.42 times and 1.19 times that of wide field microscope, respectively, and the fast 3D high-resolution optical sectioning imaging of biological samples is realized, which lays a technical foundation for further developing the rapid high-resolution imaging of the whole cells and tissues in vivo.