The effectiveness of several vaccines against coronavirus disease (COVID-19) has been reported in the real-world setting. However, it is still unknown how long antibodies persist following vaccination and whether or not the persistence of antibodies has a protective effect against COVID-19. Healthcare workers who had received two doses of the BNT162b2 mRNA COVID-19 vaccine were enrolled, and a single-center study was conducted at the National Hospital Organization Hakodate National Hospital. Serum samples from all participants were collected 13–21 weeks (median: 20 weeks) after the second dose of vaccination. The antibody titers were measured using an electrochemiluminescence immunoassay (Elecsys® Anti-SARS-CoV-2 S). Data on characteristics of the participants were gathered from patient records and interview sheets. A total of 401 participants, among whom 70.1% were women and the median age was 42 years, were evaluated in this study. None of the participants had a definite COVID-19 history, and all participants who received complete vaccination showed positive antibody titers. The antibody titer was observed to be higher in participants with younger age (p < 0.001) and those who were females (p = 0.028). Despite the higher risk of infection than that of the general public, no vaccinated staff developed breakthrough infections. This study demonstrates the significant contribution of the BNT162b2 vaccine in the acquisition of anti-SARS-CoV-2S antibodies; therefore, the general population should benefit from these two vaccine doses, which are expected to be protective for at least five months.