Robot-assisted brain tissue biopsy plays a pivotal role in diagnosing complex intracranial lesions. This study retrospectively examines the factors influencing the diagnostic yield and target point error (TPE) in frameless intracranial stereotactic brain biopsy using the Remebot surgical robot. The clinical data of 584 patients who underwent robot-assisted stereotactic brain biopsy at Xuanwu Hospital, China, between April 2019 and December 2023 were analysed. The data collected included histopathological diagnosis results and TPEs. Univariate and multivariate logistic regression analyses were performed to explore the factors influencing diagnostic yield. Multivariate linear regression was used to analyse the factors influencing TPE. MRI enhancement was an independent factor influencing the diagnostic yield, with enhanced lesions showing a higher rate (OR = 0.017, 95% CI: 0.007-0.041; P < 0.001). Larger target volumes were also correlated with higher positivity rates (OR = 1.484, 95% CI: 1.259-1.751; P < 0.001). Smaller TPEs were associated with higher positivity rates (OR = 0.103, 95% CI: 0.045-0.233; P < 0.001). Larger entry point tangential angles resulted in smaller TPEs (b = -0.093, t = -38.034, P < 0.001), and supratentorial locations had smaller errors than infratentorial locations did (b = -0.514, t = -9.182, P < 0.001). Markpoint registration was more accurate than facial laser scanning registration was (b = -0.064, t = -2.484, P = 0.013). Robot-assisted frameless stereotactic biopsy is an accurate and safe method. It is crucial to strictly adhere to the surgical indications, such as selecting cases with significant imaging enhancement. The puncture path should be planned rationally, with the biopsy needle inserted as vertically as possible to the cranial surface. A more suitable registration method should be chosen, and if higher precision is required, marked point registration should be used. This will ensure the accuracy of the target and achieve a higher diagnostic yield.