To improve voluntary donation and public health in the face of Pakistan's shifting regulations and challenges, it is crucial to comprehend the blood donation patterns of undergraduate students in Peshawar. Objective: To compare and assess undergraduate students in medical, allied health sciences, and non-medical fields in Peshawar, Pakistan, on their knowledge, attitudes, barriers to, and motivations for, blood donation. Methods: An eight-month observational cross-sectional study with 1232 undergraduate students from various academic fields was conducted. Participants were assessed through a comprehensive questionnaire regarding blood donation. Data were analyzed using SPSS version 27.0, employing descriptive statistics and the chi-square test. Results: The study enrolled 1232 undergraduate students, with 65.2% male and 34.6% female participants. Among them, 82.3% were aware of their blood group. While 24.74% exhibited poor knowledge about blood donation, 62.98% demonstrated moderate knowledge, and 12.29% had good knowledge. Additionally, 39.9% had never donated blood, but 83.9% expressed willingness to donate in the future. Fear of needles (13.90%), fear of infection (13.20%), and medical unfitness (13.10%) were significant barriers, while motivations included saving lives (22.20%), helping friends and family (19.40%), and moral obligation (14.80%). Conclusions: The study concluded that undergraduate students in Peshawar possess a moderate level of understanding regarding blood donation, with medical students showing higher knowledge levels. Despite variations across disciplines, positive attitudes towards donation were common, indicating potential for increased voluntary donation