In order to improve the accuracy of in-camera parameter calibration and reduce the error caused by inaccurate estimation of corner coordinates, a subpixel iterative corner optimization algorithm was proposed. By modeling the camera image, integrating the distortion mathematical model, and using the subpixel iterative algorithm, the gradient value changes of points in the search window were calculated and the corner coordinates were iteratively optimized to provide more accurate initial values for calibration. Combining with the optimized corner coordinates, Zhang's calibration method was used to solve the internal parameters, and the influence of corner points on the acquisition of diagonal points and camera calibration was discussed through experiments. The calibration accuracy was characterized by reprojection error, and the effectiveness of the iterative algorithm was tested. The experimental results show that the algorithm can effectively improve the accuracy of corner acquisition and the calibration accuracy of camera parameters by using fewer iterations.