Commercial confocal microscopy usually utilizes two-dimensional galvanometer to scan specimen, the frame rate is limited within 30 f/s, and commercial confocal microscopy is 10 f/s or lower. In order to improve the imaging speed of confocal system, and meet the needs of real-time observation of in vivo imaging, in the high-speed confocal line scanning laser microscopy, the sample is illuminated by one-dimensional scanning la-ser beam. The imaging speed is greatly improved. At the same time, a slit is placed before the linear array CCD to filter out to the non-focused plane stray light and improve the image quality. Experiments show that the optical magnification is 55, horizontal resolution is higher than 2.2 μm, and the frame rate is up to 50 frame per second when the linear CCD scans with 28 kHz horizontal frequency and 512 pixels×2048 pixels image resolution, im-age experiment of plant and animal cells prove that this imaging system could be used in ex vivo or in vivo cell image.