Due to the Depth of Focus (DOF) which is tens of times more than particle diameter in digital in-line Fresnel holography system, resolution of particle axial position is low and uncertain. In order to reduce the impact of DOF,an auto-focusing algorithm for accurate location of particle was proposed based on the most gradient. In the focus window containing particle, the gradients in all possible directions were calculated. Then, all of the possible gradients were compared, and the largest gradient was chosen as the result. At the same time, in order to improve the precision and stability of auto-focusing algorithm, another threshold parameter was introduced to eliminate the interference caused by background information and noise. Through experiments, we find out that the auto-focusing algorithm has the strong single peak feature and good stability.