Objective: To preliminarily verify the accuracy of navigation-assisted seed implantation by comparing preoperative and actual differences in puncture characteristics and dosimetry in computed tomography-guided, navigation-assisted radioactive iodine-125 seed implantation using 3D-printed templates for the treatment of malignant tumors. Methods: A total of 27 tumor patients who were treated with seed implantation under combination guidance in our hospital between December 2018 to December 2019 were enrolled in this study. Navigation needles (n=1–3) were placed in each patient to obtain preoperative and intraoperative puncture information, including angle, depth, insertion point, and tip position; we also investigated the dosimetry parameters in the preoperative and postoperative plans, including D90, V100, V150, V200, minimum peripheral dose (MPD), conformal index, external index, and homogeneity index of the target area. The t-tests and nonparametric correlation tests were used for analysis (P<0.05 was considered significant). Results: The means errors of the angle, depth, insertion point, and tip position were 0.47 ± 0.521°, 0.35 ± 0.238 cm, 1.7 ± 0.99 mm, and 3.1 ± 1.75 mm, respectively. There were no significant differences between the intraoperative and preoperative angles (P = 0.271), but there was a significant difference in depth (P = 0.002). Errors of the angle, depth, and insertion point were larger for the pelvic/retroperitoneal area than for the head and neck/chest wall (P < 0.05). With the exception of MPD, there was no significant difference in dosimetry indices between the postoperative and preoperative plans (P > 0.05). The MPD in the postoperative plan was higher than that in the preoperative plan (mean: 72.1 Gy and 63.8 Gy, respectively; P < 0.05). Conclusion: Seed implantation under combination guidance showed good accuracy, and the actual intraoperative puncture information and postoperative doses were in good agreement with those in the preoperative plan, thereby demonstrating promising prospects for further development.