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Background Healthcare workers (HCW) are exposed to an increased risk of COVID-19 through direct contact with patients and patient environments. We calculated the; seroprevalence of SARS-CoV-2 in HCW at Eastern Health, a tertiary healthcare network in Victoria, and assessed associations with demographics, work location and role. Methods A cross-sectional cohort study of HCW at Eastern Health was conducted. Serum was analysed for the presence of antibodies to SARS-CoV-2, and all participants completed; an online survey collecting information on demographics, place of work, role, and exposures; to COVID-19. Seroprevalence was calculated as the proportion participants with SARS-CoV-2; antibodies out of all tested individuals. Results The crude seroprevalence of SARS-CoV-2 antibodies in this study was 2.17% (16/736). Thirteen of the 16 (81.2%) positive cases had previously been diagnosed with COVID-19 by PCR: the seroprevalence in the group not previously diagnosed with COVID by PCR was 0.42% (3/720). Having direct contact with COVID-19 patients did not increase the likelihood of having positive serology. A prior history of symptoms consistent with COVID-19 was associated with a higher likelihood of having positive serology (OR 17.2, p = 0.006, 95%CI: 2.25–131.55). Conclusion Our calculated seroprevalence of 2.17% is higher than estimated in the general Australian population, but lower than that reported in HCW internationally. The; majority of those with positive serology in our study had previously been diagnosed with COVID-19 by PCR based testing. Seropositivity was not associated with interaction with COVID-19 positive patients, highlighting effective infection prevention and control practices within the workplace.