Objective To investigate the attitudes of patients towards the donation and usage of clinical biosamples and their affecting factors. Methods Questionnaires of donation of clinical biosamples were prepared and 639 patients in Renji Hospital of Shanghai Jiao Tong University School of Medicine were randomly selected. The attitudes and motivation of patients towards donating clinical biosamples, usage of biosamples, expectations for feedback of research results, and trust in medical institutions were investigated and statistically analyzed. Results The response rate was 81.7%. And 66.7% of respondents would donate biosamples and 40.8% of them would donate anonymously; 17.2% of respondents desired that they could withdraw their donations anytime afterwards; and 42.3% of respondents trusted or strongly trusted medical institutions. The donation rate of young respondents with high education levels was high (P<0.05). The donation rate of patients with hepatitis B was significantly lower than that of other patients (26.7% vs 70.4%, P<0.05). Conclusion The willingness of Chinese patients to donate biosamples is lower than that of patients of other countries. The trust level of patients towards medical institutions and their education levels may be the main factors affecting the donation. The survey is helpful for understanding the real attitudes of patients towards biosample donation, improving their awareness of usage of and clinical research on biosamples, enhancing their willingness of biosample donation, and promoting the development of ethical laws for biobanks of China.