Background: There is a high prevalence of HBsAg in adults in Lao PDR, as seen in first-time blood donors. The high positive rate is also detected in repeat donors although the cause is unclear. By surveying blood donors, this study examines the general population’s knowledge level of HBV infection and assesses the public’s perception of vaccination. Objective: The aims of this study were to assess knowledge, attitude and practices (KAP) of blood donors regarding HBV infection. Methodology: We conducted a cross-sectional study to collect the KAP information among consenting students of high school and university in March-September, 2018. A self-answered questionnaire was also utilized among blood donors in Vientiane Capital, Luang Prabang and Khammuan Provinces. The questionnaires were distributed and completed in the classroom setting with investigators proctoring. Descriptive analysis was performed on the socio-demographic characteristics, Student’s T-test were performed to examine the difference of mean and linear regression models the association between dependent and independent variables. Results: Among 625 blood donors (132 health science students and 477 non-health science students), all had a positive attitude towards blood donation, but they had low knowledge towards hepatitis infection and prevention. Multivariable analysis showed that x factors were significantly associated with HBV vaccination. This included gender, level of education, being a health science student, HBV positivity status and awareness not to donate blood and knowledge towards vaccination. HBV vaccination hesitation was observed in blood donors who did not know that vaccination can prevent infection, and most blood donors (76.9%) answered that they were healthy so did not see the need for the vaccine and did not understand this vaccination method as prevention. A fear of injections, belief that they would not be able to drink alcohol for two weeks surrounding vaccination and that it was not necessary to prevent HBV infection were reasons given to not seek HBV vaccination. Conclusion Blood donors had overall poor knowledge on hepatitis B infection and prevention, particularly among the non-health student donors. Blood donors should be aware of and understand their HBsAg testing status and broaden their perception and knowledge on HBV infection and prevention.