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Background Antibody (Ab) tests for SARS-CoV-2 virus allows for the estimation of incidence, level of exposure and duration of immunity acquired by a previous infection. In health workers, the hospital setting might convey a greater risk of infection. Aims To describe the frequency of immunoglobulin G (IgG) Abs (IgG-Abs) to the SARS-CoV-2 virus among workers at a third-level university hospital in Colombia. Methods In this cross-sectional study, we included medical and non-medical personnel with at least one real-time polymerase chain reaction (RT–PCR)/antigen test between March 2020 and March 2021. In April 2021, an IgG-Ab test against SARS-CoV-2 was conducted for all participants and replicated 2 weeks later in a random sample (10%). The frequency of IgG-Abs is presented based on status (positive/negative) and time elapsed since RT–PCR/antigen test (<3 months, 3–6 months, >6 months). Results We included 1021 workers (80% women, median age 34 years (interquartile range 28–42), 73% medical personnel, 23% with previous positive RT–PCR/antigen). The overall seroprevalence was 35% (95% CI 31.6–37.4, 35% in medical and 33% in non-medical personnel). For those with a previous positive RT–PCR/antigen test, the seroprevalence was 90% (<3 months), 82% (3–6 months) and 48% (>6 months). In participants with a previous negative RT–PCR/antigen test, the seroprevalence was 17% (<3 months), 21% (3–6 months) and 29% (>6 months). Conclusions High IgG-Ab positivity was found in hospital personnel, regardless of work activities. The prevalence of detectable Abs differed by previous RT–PCR/antigen status and time elapsed since the diagnostic test. [ABSTRACT FROM AUTHOR]