Background: This study examined and compared the attitudes and willingness of guardians of children with cancer and adult cancer patients toward donating biospecimens and clinical data for cancer research. Methods: We conducted a cross-sectional study among guardians of children with cancer (Guardian group) from Shanghai Children's Medical Center and adult cancer patients (Adult group) from Shanghai Ninth People's Hospital between February 1, 2019, and January 31, 2020. Participants' demographic data, willingness, and motivations for biospecimen donation were collected and analyzed. Results: Of 670 participants, 90.8% (318/350) in the Guardian group and 88.1%(282/320) in the Adult group completed the questionnaire. Most participants were willing to donate residual tissue samples (92.8% in the Guardian group vs. 79.4% in the Adult group, p ψ = 0.032) and clinical data (94.0% vs. 72.3%, p ψ < 0.001) for medical research. Logistic regression analysis indicated that only child status (odds ratio [OR] = 0.140, p = 0.02), history of blood donation (OR = 4.467, p = 0.019) in the Guardian group, education (OR = 0.387, p = 0.037), and history of blood donation (OR = 2.556, p = 0.016) in the Adult group were significantly associated with participants' willingness to donate biospecimens. The primary motivation for donation was helping other patients with cancer (65.4% vs. 24.5%,