Abstract: The reconstruction algorithm of single-frame lensless microscopic imaging was investigated. The experimental setup of the lensless microscopic imaging system and the two reconstruction algorithms of L-R(Lucy-Richardson) and ASM (angle spectrum method) were introduced. Comparing the resolution of the USAF resolution plate image reconstructed by the two algorithms, according to the Rayleigh criterion, the amplitude image resolution acquired by the SSM is up to 3.10 μm and the calculation time is the least (0.9 s), which proves ASM is the best reconstruction algorithm of single-frame lensless microscope imaging. In addition, the cell imaging experiments were performed using a lensless microscopic imaging system combined with ASM for image reconstruction. Results show that the lensless imaging field of view is 4.4 times than that of a 5^× microscope, and its resolution is between 5^× and 10^× optical microscopes. The statistical advantage is obvious, and it has broad application prospects in the biomedical field.