Objective: To explore students’ mastery condition of reproductive health knowledge and its influencing factors under the new media environment, in order to provide evidence for educators and administrators to implement targeted education. Methods: A cluster random sampling method was used, and students from two medical universities and two comprehensive universities in Nanjing were selected to accept a questionnaire survey of reproductive health knowledge. Results: The average score of reproductive health knowledge was 23.64 ± 5.95. As the answer of “Will women be pregnant when they lack sexual arousal during sexual intercourse”, 61.5% of students chose “possible”, 3.6% chose “impossible”, and 34.9% chose “unknown”. As contraceptive methods, the awareness of condom was the highest, up to 91.0%, followed by 87.5% of oral contraceptives, and of subdermal implants, as low as 17.0%. There were significant differences in levels of reproductive health knowledge between children with and without siblings, and between different sexes. Conclusion: College students partly grasp the knowledge of reproductive health, but not systematically or comprehensively. Reproductive health education could be carried out through various ways, especially the network, which is college students’ favorite platform.