The particle fields is generally measured by digital in-line holographic technique. But the resolution of particle axial position is lower due to the impact of zero-order image, twin-image noise and the depth of focus(DOF). Generally, DOF is tens of times as many as particle diameter. The large DOF has been a vexing problem in extraction of particle position from digital in-line holograms. DOF is affected by some recording parameters, i.e. NA of recording system, size of CCD, recording distance and the wavelength. Resolution of particle axial position can be improved by reducing depth of focus and recording distance. In this paper, an auto-focusing algorithm based on the most gradient for accurate location of particle by selecting proper window in accordance with particle size is proposed. At present, there are a lot of auto-focusing algorithms based on gradient, which calculate the gradient value only in appointed fixed directions, but the real gradient direction may not be the appointed direction. In order to solve the problem, an auto-focusing algorithm based on the most gradient and threshold is proposed. The gradients in all possible directions are calculated, then compare all of the possible gradients, and choose the largest gradient as the result. At the same time, in order to improve the precision and stability of auto-focusing algorithm, another threshold parameter is introduced. Through simulations and experiments, the algorithm has the characteristics such as strong single peak feature and good stability, which could decrease the impact of DOF to resolution of particle axial position.