Objective To evaluate the application value of intraocular biopsy in the diagnosis of atypical intraocular lesions. Methods The clinical data of 31 patients (31 eyes) with atypical intraocular lesions were retrospectively analyzed. All patients received intraocular biopsy including anterior chamber puncture, vitreous puncture and vitreous biopsy followed by pathological cell examination. Cytological examination was immediately performed for all biopsy fluids or tissues; biopsy times, the positive detecting rate and independent pathological diagnosis rate were analyzed. Intraoperative and postoperative complications were observed. Eyeballs with biopsy-suggested malignancy lesions were enucleated and underwent histopathological analysis. The biopsy results and histopathological results were compared and analyzed.Result Thirty-one eyes received 35 times of biopsy operation in total. The available samples harvested from 29 patients through 31 operations were valid for pathological cell examination，the positive detecting rate was 88.6%. Among the 31 eyes, 12 eyes had malignant lesions; 15 eyes had benign lesions; two eyes were diagnosed with benign lesions initially, but corrected to malignant through the second biopsy;the lesions in two eyes were not determined by biopsy. Among the 29 eyes with valid biopsy, 23 eyes were diagnosed independently by pathological examination; the diagnosis of the other six eyes was made based on pathological examination and clinical features. The independent pathological diagnosis rate was 71.4%. The complications included intraocular bleeding in five eyes, retinal detachment in three eyes and more serous inflammation in one eye. The sensitivity for diagnosis of malignant lesions was 85.7% and the specificity was 100.0%. The predictive value of positive test was 100.0% and the negative one was 86.7%.Conclusion Intraocular biopsy has important values in the diagnosis of atypical intraocular lesions.