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Image-guided surgery (IGS) can reduce the risk of tissue damage and improve the accuracy and targeting of lesions by increasing the surgery’s visual field. Three-dimensional (3D) medical images can provide spatial location information to determine the location of lesions and plan the operation process. For real-time tracking and adjusting the spatial position of surgical instruments, two-dimensional (2D) images provide real-time intraoperative information. In this experiment, 2D/3D medical image registration algorithm based on the gray level is studied, and the registration based on normalized cross-correlation is realized. The Gaussian Laplacian second-order differential operator is introduced as a new similarity measure to increase edge information and internal detail information to solve single information and small convergence regions of the normalized cross-correlation algorithm. The multiresolution strategy improves the registration accuracy and efficiency to solve the low efficiency of the normalized cross-correlation algorithm.