The national hepatitis B vaccination program in Taiwan began in July 1984. The purpose of this repeated survey was to investigate the levels of anti-HBs (hepatitis B surface antibody) in the first-year students of a 5-year nursing program in Taiwan. Each year during 2000–2006, the entering students of the 5-year nursing program at Fooyin University, a vocational university located in southern Taiwan, were examined for their HBsAg and anti-HBs status using commercially available microparticle enzyme immunoassay. The seroprevalence of HBsAg (+) showed a significant trend of decrease, dropping 57% from 4.9% in 2000 to 2.1% in 2006. The seroprevalence of anti-HBs (+) also showed a significant trend of decrease, dropping 49% from 77.1% in 2000 to only 39.7% in 2006. With the relatively low seroprevalence of anti-HBs (+) of the future healthcare workers and high HBV endemicity in Taiwan, recommendation of serology test before boosting to nursing students before they proceed their clinical practice is prudent.