COVID-19 infection caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to affect people as a global threat, and the number of cases is increasing every day. Healthcare workers who face potential COVID-19 exposure are at high risk of SARS-CoV-2 transmission. Estimating the prevalence of infection among healthcare professionals, determining the related risk factors and applying effective infection control measures are essential for the continuity of the health system. The aim of this study was to investigate the seroprevalence of SARS-CoV-2 among healthcare workers in our hospital who have participated extensively in the monitoring of COVID-19 patients. In the study, the anti-SARS-CoV-2 IgG antibody test results of 774 healthcare workers between March 24, 2020, and September 10, 2020 were analyzed retrospectively. Age, sex, profession, and the status of being diagnosed with COVID-19 before the antibody test were determined for the healthcare workers in the study. When the anti-SARS-CoV-2 IgG antibody results were evaluated, it was determined that 57 healthcare workers were positive, 708 healthcare workers were negative, and 9 healthcare workers were borderline. The seroprevalence among the workers of our hospital was found to be 7.4%. The antibody positivity rate was 75.6% in individuals diagnosed with COVID-19 by SARS-CoV-2 PCR (polymerase chain reaction) and/or thoracic computed tomography and it was found to be 3.5% in individuals without the diagnosis. The semi-quantitative antibody index values of the healthcare workers who were seropositive and diagnosed with COVID-19 before the test (n= 31) and those who did not (n= 26) were statistically compared and a significant difference was found between the two groups (p< 0.01). In our study, the highest seropositivity was observed among residents (12.3%) and among nurses (11.1%), respectively. When the seropositivity rates of the residents and the nurses were compared with other occupational groups, the differences were found to be statistically significant (p= 0.04, p= 0.04, respectively). In conclusion, the seroprevalence of SARS-CoV-2 was determined as 7.4% among healthcare workers in a tertiary hospital with high patient admissions during the COVID-19 pandemic. Considering that SARS-CoV-2 seroprevalence was announced as 0.81% in the press release made by the Ministry of Health of Turkey in July 2020, it is seen that the rate of seroprevalence among health care workers is significantly larger than the community. Determination of the seroprevalence in the general population and large-scale studies are needed for risk assessment in healthcare professionals.