The World Health Organization estimated that around 66 thousand HBV are caused by needlestick injuries annually. Healthcare students should be aware of HBV transmission route and preventive measures. This study assessed the knowledge, attitudes, and practices toward HBV among Jordanian healthcare students and its associated factor. A cross-national study conducted from March to August. The participants were asked to complete the questionnaire. It had four sections: participants' sociodemographic, knowledge, attitudes, and practices about HBV. 2322 participants were enrolled, 67.9% were females, 26.4% were medical student, 35.9% were in the 3rd year. 40% of the participants held a high level of knowledge and attitude. 63.9% of participants had good practices toward HBV. Medical students in the last year of study, encountered HBV patients , had better knowledge. Male students from medicine college, encountered HBV patients and had extra HBV courses showed better attitude. High practice level was associated with being dentistry student, at 5th year, encountered HBV patient and extras HBV courses. This study demonstrated insufficient knowledge and attitudes toward HBV, the practices level toward HBV among healthcare students was promising. Subsequently, public health efforts should modify the knowledge and attitude gaps to reinforce awareness and minimizing risks of the infection.