To assess the measles seroepidemiology of healthcare workers (HCWs), from 2004 to 2009 all newly recruited staff at a university hospital in Taiwan, were tested for specific immunoglobulin G antibodies for measles virus using enzyme-linked immunosorbent assays. A total of 1584 HCWs were included. The positivity rate of measles antibody for physicians and nurses was about 90%. The positivity of measles antibody in different age groups was 78.1%, 93.9% and 94.2% for 20–29, 30–39 and ≥40 years age groups, respectively. Equivocal results were more frequent in the 20–29 (17.1%) years age group. A lack of antibody against measles was reported for 66 (4.2%) of those tested. Medical student (8.3%) and housekeeping personnel (5.3%) had the greatest lack of antibodies. This study demonstrates the waning measles immunity in younger HCWs in Taiwan, which poses the potential risk of nosocomial transmission of measles. Periodic surveillance and revaccination of susceptible HCWs is recommended.