Abstract: Sperm motility performance is an important basis for evaluating sperm motility.At present,the relatively advanced computer-assisted sperm analysis （CASA） method uses graphics processing technology to process and analyze sperm images taken by phase contrast microscope.The equipment is large and expensive,and the imaging field is small.More importantly,the motion trajectory obtained by this method is on the plane.In fact,sperm movement occurs in three dimensions.The key step to obtain 3D motion trajectory is 3D positioning.Therefore,a method to obtain 3D positioning of porcine sperm by lensless diffraction imaging is proposed in this study.In this method,based on the diffraction image reconstruction,image processing algorithm is used to obtain its plane coordinates,and focusing degree evaluation index and self-focusing algorithm are designed to obtain its height coordinates by using the principle that the reconstruction distance affects the contrast of virtual and real images.Furthermore,the 3D location of porcine sperm individual is realized.The results show that this method can achieve the accuracy of±1.4 μm and±10 μm in the horizontal and vertical directions respectively.This method is expected to provide more scientific data support for sperm quality detection and reproductive medicine research.