Healthcare workers are a category of subjects at risk of infections higher than general population, though the introduction of vaccination against hepatitis B generally reduced this risk. In the present research, 2137 students of graduate course for health professions were subdivide in two age classes: 25 years old or younger and older than 25 years old according to the vaccination strategy defined by the law 165/91. A high number of students (82.3%) appeared immune against hepatitis B virus, whereas 16.7% were negative. About 30% of negative subjects declared vaccination. Females and subjects 25 years old or younger showed a significantly higher prevalence of anti-HBsAg antibodies (p < 0.001) than males and older subjects respectively. The obtained results show the need to evaluate the immunity status of the healthcare workers in training with the aim to reduce the number of susceptible subjects.