Background: College students are the key group we should pay more attention for acquired immune deficiency syndrome (AIDS) prevention and control in recent years in China, and few studies of HIV non-occupational post-exposure prophylaxis(nPEP) knowledge awareness and service acceptance have been conducted among them in China. This study conducted a cross-sectional survey to understand the service acceptance of nPEP and its influencing factors among college students in the three cities of China. Methods: A questionnaire survey was conducted to collect information on socio-demographic, behavioral characteristic, HIV/AIDS knowledge, nPEP knowledge, acceptance of nPEP services among the college students in Beijing, Shenzhen, and Kunming of China from March to April of 2019. Each participant completed a computer-assisted or mobile phone-assisted self-interview with informed consent and completed an anonymous questionnaire on line. Multivariable logistic regression analyses identified predictors for service acceptance of nPEP. Results A total of 4698 students were surveyed with the average age of 20 years old. 98.0% (4605/4698) of them were undergraduates, 21.8%(1022/4698) had sexual intercourse; 48.6% (2282/4698)had heard of nPEP, among which4.95%(113/2282) received nPEP services. The rate of AIDS knowledge awareness was 85.6% (5495/4698) with the differences statistically significant between the three cities. The nPEP knowledge awareness rate was 16.5% (774/4698). There were differences in receiving nPEP services among students of different ages, genders, sexual behaviors, and knowledge of AIDS by univariate analysis. Multivariable analyses indicated that age group of 18 and under (OR=2.551, 95% CI=1.153-5.646), male (OR=3.131, 95% CI=1.866-5.253), homosexual behavior (OR=4.661,95%CI=2.658-8.172), heterosexual behavior (OR=1.676, 95% CI=1.040-2.947), no awareness of AIDS (OR=3.882, 95% CI=2.371-6.356) and nPEP (OR=4.788, 95% CI=2.50-9.169) knowledge, were associated factors with the service acceptance of nPEP among the college students. Conclusion The low acceptance of nPEP services was mainly affected by nPEP knowledge awareness among the college students and further publicity and education of nPEP knowledge are necessary, as well as dissemination of knowledge of HIV/AIDS prevention and treatment. More attention should be paid to the factors associated with acceptance of nPEP services.