More literatures regarding radiocolloid-based dynamic sentinel lymph node biopsy (DSNB) in penile cancer with clinically negative groin (cN0) have been published since previous meta-analysis in 2012. This updated meta-analysis was to assess the accuracy of the procedure in penile cancer with cN0 disease and explore its relative factors on the basis of current evidences. We performed a review of PubMed, Ovid/Embase, and the Cochrane Library in March 2016 according to the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement. Study quality was evaluated by the use of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). A random effects model was used for statistical pooling. Publication bias was evaluated by the use of funnel plot and Egger's test. Meta-regression, subgroup and sensitivity analysis were conducted to explore the sources of heterogeneity. A total of 27 articles were included. Two articles had two different cohorts and each cohort was considered a separate study. Overall 29 studies were used for sensitivity and negative predictive value (NPV) meta-analysis. The pooled sensitivity and NPV was 88 % (95 % CI 84-90 %) and 99 % (98-99 %), respectively. Meta-regression and subgroup analysis revealed that the use of preoperative ultrasonic scan (USS) ± fine-needle aspiration cytology (FNAC), surgical exploration of wound for suspicious lymph nodes (LN), immunohistochemistry (IHC) and extensive experience were significantly associated with the improved sensitivity of DSNB. Radiocolloid-based DSNB is a promising staging modality to detect inguinal micrometastasis in penile cancer without clinically positive inguinal LN. Preoperative USS ± FNAC and surgical exploration are effective supplements to exclude potentially clinical involvement, and IHC makes the diagnosis of occult metastasis in SLN more likely. The multidisciplinary and multistep procedure should be performed by skilled teams in specialized centers.