Biliary strictures are frequently encountered in clinical practice. The determination of their nature is often difficult. This study aims to systematically evaluate the efficacy and safety of the second generation of digital single-operator cholangioscopy (SpyGlass DS, DSOC) in indeterminate biliary strictures (IBDS) through biopsies. All relative studies published in Medline, the Cochrane Library, Web of Science, and EMBASE were included. The diagnostic tests for IBDS were compared to the surgical histology, autopsy, or long-term clinical follow-up. The methodological quality of the included studies was evaluated by the Quality Assessment of Studies of Diagnostic Accuracy Included in Systematic Reviews (QUADAS-2). A total of 11 studies, which involved 356 patients diagnosed through biopsies, were included. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio were 0.74 (95% CI: 0.67-0.80), 0.98 (95% CI: 0.95-1.00), 10.52 (95% CI: 5.45-20.32), 0.31 (95% CI: 0.23-0.41), and 65.18 (95% CI: 26.79-158.61), respectively. The area under the curve (AUC) was 0.9479, and the pooled adverse event rate was 7%. The sensitivity and specificity in the heterogeneity analysis were I2 = 48.1% and I2 = 25.4%, respectively. SpyGlass DS is a safe and effective technique for IBDS. However, future randomized trials are needed to determine optimal number of biopsies.