We assessed university students’ knowledge, attitude, and practice toward blood donation and identified the factors that promote or hinder their willingness to donate. We employed a multicenter cross-sectional design, collecting data from August to October 2022 through self-administered questionnaires available in Arabic and English. Both online (Google Forms) and paper surveys were utilized. Data were analyzed using R Statistical Software (v4.1.3; R Core Team 2022). A total of 12,606 university students (7966 females and 4640 males) from 16 countries completed the questionnaire; of them, 28.5% had a good knowledge level regarding blood donation, and 22.7% had donated blood at least once. Students in health science colleges had significantly more awareness of blood donation (p-value < 0.001), but there were no significant differences in practice (p-value = 0.8). Barriers to donation included not being asked (37%), medical ineligibility (33%), fear of pain or infection (18%), concerns about negative health effects (18%), difficulty accessing donation centers (15%), and medical mistrust (14%). Individuals aged > 20 years had significantly higher odds of possessing a high knowledge level (adjusted odds ratio [aOR] 1.77, p < 0.001). Private and international university enrollment was associated with increased knowledge (aOR 1.19, p-value < 0.001 and aOR 1.44, p-value = 0.003), while non-health science college students had lower odds (aOR 0.36, p < 0.001). Regarding blood donation status, participants > 20 years old were more likely to donate (aOR 2.21, p < 0.001). Conversely, being female, having congenital or chronic diseases, and possessing low knowledge levels were associated with decreased odds of blood donation (all p < 0.05). University students show insufficient knowledge about blood donation, with health science students displaying higher awareness levels. Despite their positive attitudes, blood donation rates remain low across all disciplines. It is imperative to enhance education and accessibility to foster a culture of blood donation among students.