Background and purpose To evaluate the application of treatment planning system (TPS) assisted large-aperture computed tomography (CT) simulator in percutaneous biopsy, and report our initial experience of the accuracy and safety of this procedure.Methods From November 2018 to December 2019, treatment planning system assisted large-aperture CT simulator guided percutaneous biopsy was performed on 38 cases, with 34 of percutaneous lung biopsy, three of abdominal lesions biopsy, and one case of deep supraclavicular lymph node biopsy. The major results including planned and actual puncture parameters, the success rate, pathological information and complications were recorded. The analyses of puncture accuracy were accomplished by pared-t test and Wilcoxon rank sum test. And the risk factors of puncture accuracy and complications were further identified.Results The entire cohort achieved one-time success of biopsy. No significant differences were presented between planned and actual puncture depth and direction (P = 0.436 and 0.382), indicating the precision of the process. And the pulmonary puncture location was related to the accuracy of puncture direction (P = 0.033). Biopsy specimens were successfully obtained in 38 cases. The diagnostic rate of malignancy was 76%, of which 80% for initial treatment group and 69% for treated group, respectively. For patients with pulmonary biopsy, 12 had minor pneumothorax and 2 progressed to massive pneumothorax. Only three cases suffered needle track bleeding, and no other complications were observed. Additionally, the regression analysis found a significant correlation between puncture angle and the incidence of pneumothorax (P = 0.027).Conclusions TPS assisted large-aperture CT simulator improved the procedure of percutaneous biopsy by combining the advantages of radiotherapy specialty. The initial results suggested the increase of puncture accuracy and success rate, with satisfactory safety simultaneously. It might offer new insights into the field of CT-guided percutaneous biopsy.