Background: The present study aimed to determine the prevalence of HBsAg in Health Care Workers (HCWs) in Eastern Mediterranean Region Office (EMRO) and Middle Eastern countries from 2000 to 2016. Methods: In a meta-analysis study, the databases of PubMed, ISI, Ovid, Scopus, Google Scholar, and Persian databases were searched for relevant articles on the prevalence of HBsAg in HCWs in EMRO and Middle Eastern countries. Homogeneity was assessed based on Cochran's Q-test results. Results: A total of 43 articles (110,179 people) were included. The pooled prevalence of HBsAg in HCWs of EMRO and Middle East countries was found 2.77% (95%CI: 2.64-2.83). The specific prevalence of HBsAg was 2.84% (95% CI: 2.6-3.11) in EMRO and 2.22% (95%CI: 2.13-2.31) in Middle Eastern countries. The highest and lowest prevalence rates of HBsAg among HCWs for countries with more than one study were 6.85% (95% CI: 5.74%–8.16%) in Sudan and 1.00% (95% CI: 0.94%–1.07%) in Turkey, respectively. The trends of HBsAg prevalence among HCWs decreased from 2000 to 2016. Conclusions: Based on the World Health Organization classification of HBV prevalence, intermediate HBsAg prevalence rates were detected in HCWs of EMRO and Middle East countries during 2000–2016.