Objective To explore the application of digital planning system combined with 3D-printing puncture template in the training of puncture skills for refresher doctors. Methods A total of 42 trainees who received advanced training in the Department of Radiation Oncology of Peking University Third Hospital from December 2021 to June 2022 were selected. They were randomly divided into the study group and the control group. The trainees in the study group used the teaching method of planning system combined with puncture template, while the students in the control group used the traditional teaching method. To evaluate the training outcomes by examination and questionnaire. Data were analyzed by χ² test and independent sample t test. Results The theoretical knowledge, image reading ability, and clinical skill assessment scores of the physicians in the study group were higher than those in the control group [(92.24±2.51) score compared to (90.33±3.10) score, (92.33±3.34) score compared to (87.48±4.67) score, and (90.95±2.56) score compared to (85.81±3.70) score, respectively]. The differences were statistically significant (all P<0.05). In the study group, three physicians (14.3%) believed that the learning difficulty was relatively high, compared to eleven physicians (52.4%) in the control group; furthermore, 17 individuals (81.0%) in the study group and 9 (42.9%) in the control group believed they had achieved their further education goals; after 3 months of further education, 14 individuals (66.7%) in the study group and 6 (28.6%) in the control group underwent puncture techniques. The differences were statistically significant (all P<0.05). Conclusions The teaching method of planning system combined with 3D printing template is helpful to improve trainees′ understanding of theoretical knowledge and mastering of practice skills, and it is also beneficial for cultivating relevant talents.