The presence of lymph node metastasis is the most important prognostic indicator for patients with penile cancer. However, predicting which clinically node negative patients will harbor lymph node metastases remains unclear. The aim of this systematic review is to provide an overview of biomarkers p53, Ki-67, and SCCAg in predicting lymph node metastasis (LNM) and cancer-specific survival (CSS) in penile squamous cell carcinoma (SCC). MEDLINE, EMBASE, Cochrane Library, Scopus, and ClinicalTrials.gov were searched from inception until 15 October 2020. Eligible studies were identified by three independent reviewers. Outcome measures included the presence of penile LNM and CSS. Extracted data were narratively synthesized with GRADE criteria utilized to evaluate the quality of evidence. In total, 999 articles were screened with 20 selected for inclusion. Studies reporting the use of p53 to predict LNM and CSS were rated as having the highest quality of evidence using the GRADE criteria, and the majority showed a positive association between p53 expression and LNM and CSS. All biomarkers and outcome combinations had at least one study showing a significant effect on predicting the outcome. However, studies were heterogeneous, and many reported nonsignificant effects. Identifying p53 overexpression may help one to identify patients at higher risks of LNM to be considered for early inguinal lymphadenectomy. There is contradictory and unreliable evidence for the prognostic value of Ki-67 and SCCAg in penile SCC for LNM and CSS. Larger studies are required with more rigorous methods and reports to improve the evidence base