Healthcare staff are at high risk of occupational exposure to Hepatitis B and other blood-borne diseases. Lack of education about the knowledge of Hepatitis B virus contributes to an increase in cases. This study aims to determine the knowledge of the Hepatitis B virus among the medical professionals in Duhok province, Kurdistan Region of Iraq, and to determine their knowledge of the importance of vaccination. This cross-sectional study was conducted in Duhok province, Kurdistan Region of Iraq, among medical science students from November 2022 to February 2023 and a total of 511 students participated in the study. A Self-administered questionnaire comprising 22 items categorized into five sections was distributed to the students either electronically or by paper and pen method. The survey utilized a Five-point Likert scale when assessing respondents' opinions on knowledge, attitude, and practice (KAP). Microsoft Excel and GraphPad Prism 9 were used for statistical analysis. A total of 511 responses were collected from medical, dental, pharmacy, and laboratory students. The average age of the participants was 20.74 ±1.43 years. Among the respondents, only 96 (18.8%) were fully vaccinated against the Hepatitis B virus (received 3 or more doses of the vaccine), while 294 (57.5%) were not vaccinated. Lack of vaccination programs was the major reason for not receiving a vaccination (n = 182, 62%). About 286 (55.96%) of the participants had good knowledge, attitude, and practice on Hepatitis B, manifesting median scores of 26, 18, and 20, respectively. In our study, half of the students were found to be unvaccinated, mainly due to the absence of vaccination programs. Vaccinated students exhibited better knowledge, attitude, and practice toward the infection than non-vaccinated students. Therefore, we recommend the implementation of a vaccination program as well as training on infection prevention guidelines to increase awareness and encourage vaccination.