BACKGROUND: Needlestick injuries (NSIs) are recognised as a major occupational health concern for health care workers, particularly dental assistants who frequently handle sharp instruments. However, little attention has been paid to this vulnerable group. Therefore, it is important to evaluate the prevalence of NSIs among dental assistants and identify associated risk factors to promote the safety level of dental assistants and the development of prevention strategies. METHODS: We followed PRISMA guidelines and registered the protocol with PROSPERO (CRD 42023404766), searching PubMed, Web of Science, Embase, Scopus, and Ovid databases for studies published between January 2000 and January 2023. We selected relevant studies by screening titles and abstracts, and then evaluated the full texts. Risk of bias was assessed using the Critical Appraisal Checklist for prevalence studies proposed by the Joanna Briggs Institute (JBI). RESULTS: Eleven studies involving 2663 dental assistants were included. The pooled NSIs prevalence was 44% (95% confidence interval (CI) = 0.29-0.59), with significant heterogeneity (I CONCLUSIONS: The 44% prevalence of NSIs among dental assistants implies a non-negligible risk. Instrument cleaning and the handling of local anaesthetic syringes are the principal procedures and associated factors contributing to their NSI exposure. REGISTRATION: International Prospective Register of Systematic Reviews (CRD 42023404766).