Healthcare workers (HCWs) are at the front line of the ongoing coronavirus 2019 (COVID-19) pandemic. Comprehensive evaluation of the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) among HCWs in a large healthcare system could help to identify the impact of epidemiological factors and the presence of symptoms on the immune response to the infection over time. To determine the seroprevalence of SARS-CoV-2-specific antibodies among HCWs, identify associated epidemiological factors and study antibody kinetics. A longitudinal evaluation of the seroprevalence and epidemiology of SARS-CoV-2-specific antibodies was undertaken in approximately 30,000 HCWs in the largest healthcare system in Connecticut, USA. At baseline, the prevalence of SARS-CoV-2 antibody among 6863 HCWs was 6.3% 95% confidence interval (CI) 5.7–6.9%, and was highest among patient care support (16.7%), medical assistants (9.1%) and nurses (8.2%), and lower for physicians (3.8%) and advanced practice providers (4.5%). Seroprevalence was significantly higher among African Americans odds ratio (OR) 3.26 compared with Caucasians, 95% CI 1.77–5.99, in participants with at least one symptom of COVID-19 (OR 3.00, 95% CI 1.92–4.68), and in those reporting prior quarantine (OR 3.83, 95% CI 2.57–5.70). No symptoms were reported in 24% of seropositive participants. Among the 47% of participants who returned for a follow-up serological test, the seroreversion rate was 39.5% and the seroconversion rate was 2.2%. The incidence of re-infection in the seropositive group was zero. Although there is a decline in the immunoglobulin G antibody signal over time, 60.5% of seropositive HCWs had maintained their seroconversion status after a median of 5.5 months.