Objective To evaluate the deviation between actual and simulated screw placement after cervical pedicle screw placement assisted by 3D printed navigation template, and analyze the correlation between screw placement deviation and navigation pipe length. Methods A total of 40 patients undergoing cervical 1-7 pedicle screw insertion assisted by 3D printed navigation template in Zigong Fourth People’s Hospital between February 2018 and August 2020 were included in this prospective study. These patients were divided into 3 groups randomly, including 12 patients with a 5-mm pipe length (5 mm group), 13 patients with a 10-mm pipe length (10 mm group), and 15 patients with a 15-mm pipe length (15 mm group). Three-dimensional modeling was performed on preoperative cervical CT images of these patients and simulated pedicle screw was placed. Individualized pedicle screw navigation templates were designed according to the position and direction of simulated pedicle screws, and 3D printing was performed on the cervical model and navigation templates. Preoperative 3D printed model and navigation templates were used to simulate the surgical process to confirm the safety of screws. During the operation, pedicle screw placement was performed according to the preoperative design and simulated surgical process. The postoperative CT images were registered with the preoperative CT images in 3D model. The safety of screw placement was evaluated by the postoperative screw placement Grade, and the accuracy of screw placement was evaluated by measuring the deviation of screw placement point and the deviation of screw placement direction in horizontal plane (inclination angle) and sagittal plane (head inclination angle). The influence of different navigation pipe lengths on the safety and accuracy of screw placement was analyzed. Results A total of 164 pedicle screws were inserted with navigation template assistance, including 48 screws (38 in Grade 0 and 10 in Grade 1) in the 5 mm group, 52 screws in the 10 mm group (all in Grade 0), and 64 screws (52 in Grade 0 and 12 in Grade 1) in the 15 mm group, and the difference in the grade among the three groups was statistically significant (P<0.05). When the navigation pipe length was 5, 10, and 15 mm, respectively, the screw entry point deviation was (1.87±0.63), (1.44±0.63), and (1.66±0.54) mm, respectively, the inclination angle deviation was (2.72±0.25), (0.90±0.21), and (1.84±0.35)°, respectively, and the head inclination angle deviation was (8.63±1.83), (7.15±1.38), and (8.24±1.52)°, respectively. The deviations in the 10 mm group were all significantly less than those in the other two groups (P<0.05). Conclusions In the cervical pedicle screw placement assisted by navigation template, all the screws were Grade 0 or Grade 1, with high safety. The mean deviation of the screw entry point is within 2 mm, with high accuracy. When the length of navigation pipe is 10 mm, the safety and accuracy of screw placement can be fully guaranteed.