Medical image-guided percutaneous renal puncture is an important technique to establish surgical channels in the percutaneous nephrolithotomy. High quality and real-time medical image can improve the accuracy of intraoperative puncture and reduce surgical risks. Aiming at the navigation and positioning problem of percutaneous renal puncture under free breathing, a renal puncture positioning method based on the intraoperative 2D ultrasound and the preoperative 3D CT image is introduced. In the first step, a real-time registration method was applied to the 2D ultrasound and the preoperative 3D CT image to realize rapid registration and fusion. Then the fusion image is registered in the surgical space through ultrasound probe calibration and spatial coordinate transformation, which realized the real-time positioning of puncture targets. Finally, the human abdomen model was used for the experiments, and the results showed that the root mean square error of the ultrasound probe calibration is 0.998 mm. The registration error of 2D ultrasound and 3D CT images is 0.709 mm, the average registration time was 1.15 s, and the final average positioning error is 2.265 mm.