Data on humoral and cellular responses to BNT162b2 as a booster dose, following two doses of ChAdOx1 nCov-19 vaccine, have seldom been reported. The aim of this study was to assess the positivity rates of three representative antibody assays targeting total, IgG, and neutralizing antibodies, and an interferon-γ release assay (IGRA), and to determine the longitudinal changes in quantitative antibody titers after each vaccination. A total of 1027 samples were collected from healthcare workers. The number of participants after the booster dose was 153, and they all completed a questionnaire on adverse reactions. All antibody assays showed 100.0% positivity at 1 month after booster vaccination. The median antibody titers of the assays were significantly increased compared with those after the second dose (22.1-fold increase for Roche total antibody, 14.0-fold increase for Abbott IgG, and 1.1-fold increase (97.5% inhibition) for GenScript neutralizing antibody). Cellular responses determined using the IGRA were positive in 92.8% of the participants. Most participants (72.5%) reported mild adverse reactions. Correlations between the three antibody assays and IGRA were weak or negligible, indicating a difference between humoral and cellular responses. Overall, our study provides information about booster vaccine strategies and laboratory settings, which could subsequently contribute to the control of the spread of coronavirus disease 2019.