Background: Blood donation is crucial in saving lives, as blood cannot be manufactured artificially and can thus be obtained only from human blood sources. Voluntary blood donation is the cornerstone of a safe and adequate supply of blood and blood components. We aim to understand the level of knowledge, attitude, and practice of blood donation and associated factors among undergraduate students in Nigeria. Method: We conducted a cross-sectional descriptive study among 300 undergraduate students at Enugu State University College of Medicine Parklane, Enugu, Nigeria in June 2022 to access their knowledge, attitude, and practice of blood donation. Data were analyzed using multivariable logistics regression to identify factors associated with blood donation practice. Results: Most of the students had good knowledge of blood donation, and it was revealed that the initial source of information was mostly from school (37.7%), 89.0% of participants knew their blood group, and 98.3% were aware of infections through blood donation. Also, the attitude of our respondents towards blood donation revealed that the majority (95.0%) agreed blood donation saves lives, that blood donation is good (91.0%), and that voluntary blood donation (84.7%) is the best source of donor blood; most were indifferent to paid donation. Regression analysis suggests that knowing own blood group, having a positive attitude towards blood donation, and being willing to donate in the future were significantly associated with blood donation practice.  Conclusion: Hence, there is a need for adequate enlightenment campaigns and health education on the need for voluntary blood donation to improve knowledge of, change attitudes towards voluntary blood donation, and ultimately encourage undergraduate students in developing countries to donate blood.