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Abstract Background This study aimed to investigate the specific risk factors for the transmission of novel coronavirus (SARS-CoV-2) among healthcare workers in different campuses of a university hospital and to reveal the risk factors for antibody positivity. Methods In this retrospective cross-sectional study, 2988 (82%) of 3620 healthcare workers in a university hospital participated. The coronavirus disease 2019 (COVID-19) antibody was investigated using serum from healthcare workers who underwent COVID-19 antibody testing. The antibody test results of the participants were evaluated based on their work campus, their profession and their workplace. The statistical significance level was p < 0.05 in all analyses. Results Of the participants in this study, 108 (3.6%) were antibody positive, and 2880 (96.4%) were negative. Antibody positivity rates were greater in nurses compared with other healthcare workers (p < 0.001). Regarding workplace, antibody positivity was greater in those working in intensive care compared to those working in other locations (p < 0.001). Conclusions Healthcare workers are at the highest risk of being infected with COVID-19. Those who have a higher risk of infection among healthcare workers and those working in high-risk areas should be vaccinated early and use personal protective equipment during the pandemic. Trial Registration: Retrospective permission was obtained from both the local ethics committee and the Turkish Ministry of Health for this study (IRB No:71522473/050.01.04/370, Date: 05.20.2020).