Objective The aim of this study was to compare the efficacy, safety, and duration of Remebot robot-assisted frameless brain biopsy with those of standard frame-based stereotactic biopsy. Patients and methods A retrospective analysis of 66 patients undergoing stereotactic brain biopsy in our department from January 2015 to January 2019 was performed. We divided the patients into two groups: the frame-based group (n = 35) and the Remebot robot group (n = 31). Data on clinical characteristics, total procedure length, overall discomfort, diagnostic yield, complications, and postoperative length of hospital stay were retrospectively reviewed and compared between these two groups. Results No significant difference in diagnostic yield was detected in the two groups, with frame-based biopsy having a diagnostic yield of 91.4% and Remebot robot-assisted frameless brain biopsy having a diagnostic yield of 93.5%. The duration of the total procedure was 116.5 min for the frame-based biopsy and 80.1 min for the Remebot robot-assisted frameless brain biopsy (p < 0.001). There were no statistically significant differences in complication rate or postoperative duration of hospitalization between the two groups. The overall patient discomfort in the frame-based group was significantly greater than that in the Remebot robot group (visual analog scale score 2.7 +/- 1.2 versus 1.5 +/- 0.7,p = 0.001). Conclusions Remebot robot-assisted frameless brain biopsy was as efficacious and safe as standard stereotactic frame-based biopsy. However, frameless biopsy can alleviate the suffering of the patient and reduce the total duration of the procedure. Remebot robot-assisted frameless brain biopsy is easy to use and better accepted by patients than frame-based biopsy.