Background Donor milk is the preferred substitute to mother’s own milk, which can protect high-risk infants from many complications. Several milk banks have been established in China. However, the small number of donors and insufficient milk donated have become the primary obstacles for these milk banks. Research aims To explore the level of, and the factors influencing, knowledge and attitude about donor milk among currently lactating women across mainland China and to provide a reference for the development of Chinese milk banks. Methods A brief, self-reporting, prospective, cross-sectional, online survey was conducted in 2018 using The Knowledge and Attitude Questionnaire of Human Donor Milk for Hospitalized Women. Lactating women aged >18 years and willing to consent to study participation were recruited throughout China. Results All completed questionnaires ( N = 489) were included. 40.1% ( n = 196) of the participants had heard of donor milk or milk banks, and 76.7% ( n = 375) were willing to donate their milk. In the multiple linear regression analysis, educational level, cohabitation status, parity, history of delivering preterm or low-birth-weight infants, education provided by health professionals, and infants’ gestational age, weight, and feeding patterns were associated with the participants’ knowledge about donor milk ( F = 12.387, p < .001). The parity, delivery mode, mothers’ milk production, history of delivering preterm or low-birth-weight infants, education provided by health professionals, and infants’ age were associated with their attitude ( F = 9.420, p < .001). Conclusion The participants’ attitudes about donor milk were positive. However, there were gaps in their knowledge about donor milk.