Abstract: The existing spatial mismatch calibration methods can only achieve pixel-level calibration accuracy due to the limitation of principle, and are easily disturbed by the environmental noise. In this paper, a spatial mismatch calibration method for a sub-pixel-level simultaneous slightly off-axis digital holographic microscope system is proposed. In the modeling, the method not only analyzes the lateral position error caused by the image segmentation, but also further considers the longitudinal position error caused by the sensor tilt, and summarizes the calibration process as a nonlinear multi-variable optimization problem. In this paper, the particle swarm optimization algorithm is used to solve this optimization problem because of its simple structure, high convergence efficiency, and strong global search ability. In the calibration process, a phase-only wavefront based on the phase aberration is established, and the root mean square error of the phase-only wavefront is used as the target function to remove the influence of noise on the calibration accuracy. Simulation results show that the proposed method has sub-pixel accuracy, and experiment demonstrates the effectiveness of the method in the practical systems.