Objective To explore the safety and effectiveness of Remebot robot-assisted stereotactic biopsy to the diagnosis of intracranial lesions, and provide an analysis of factors affecting the diagnostic yield. Methods A retrospective study was conducted of 89 patients undergoing Remebot robot-assisted stereotactic biopsy at our institution from Oct 2016 to Dec 2019. Diagnostic accuracy, surgical complications and clinical characteristics associated with non-diagnostic biopsy were evaluated. Results Biopsy was successfully performed in all patients and a definitive histologic diagnosis was established in 81 cases with a diagnostic yield of 91%. Postoperative complication occurred in 5 patients with a complication rate of 5.6%. Univariate analysis revealed that patient's age older than 40, the large tumor size and the enhancement of lesion appeared to increase the diagnostic accuracy (all P<0.05). The size and enhancement of lesion were suggested by Logistic regression analysis to have significant influence on the diagnostic accuracy. Conclusions With advantages of minimal invasion, high diagnostic yield and low complication rate, Remebot robot-assisted stereotactic biopsy is an important medical auxiliary technology in the patients with intracranial lesions and should be worthy of further promotion. Before biopsy, more attention should be paid to the tumor size and enhancement of the lesions which significantly influence the diagnostic yield.