Healthcare workers (HCWs) are highly exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The aim of this study was to assess the seroprevalence of SARS-CoV-2 in HCWs working in a frontline hospital in Tokyo, Japan. In this observational cohort study, screening was offered to agreed HCWs, including medical, nursing, and others, as part of a mandatory health checkup. The screening test results and clinical characteristics of the participants were recorded. The antibody seroprevalence among the 4,147 participants screened from July 6 to August 21 2020, was 0.34 % (14/4,147). There was no significant difference in seroprevalence between frontline HCWs with a high exposure risk and HCWs in other settings with a low exposure risk. Of those seropositive for SARS-CoV-2, 64% (9/14) were not aware of any symptoms and had not previously been diagnosed with COVID-19. In conclusion, this study provides insights into the extent of infection and immune status in HCWs in Japan, which has a relatively low prevalence of COVID-19.