Objective: To compare the differences of common knowledge, attitudes and motivations in voluntary blood donation between students from one comprehensive university and one medical university in Nanjing. Methods: We established questionnaires via document research, expert consultation and focus group discussion. Chi-square test was performed to compare the differences of common knowledge. t-test was performed to compare the differences of attitudes and motivations. Results: Students in comprehensive university known less about blood donation than students in medical universities. The score of attitude towards blood donation in students from medical university was higher than that in students from comprehensive university. No significant differences were found between them in feelings when they are donating blood. The score of motivation of altruism in medical university students was higher than that in comprehensive university students. Among the students who did not donate blood, the score of motivation of self-efficacy and reward in medical university students was higher than that in comprehensive university students. Conclusion: It is necessary to strengthen the popularization of blood donation in comprehensive universities. Suitable broadcasting and recruiting strategies can be established according to the differences between them in motivation and attitudes to donate blood.