Objectives: The prevalence of antibodies to SARS-CoV-2 among blood donors in China remains unknown. To reveal the missing information, we investigated the seroprevalence of SARS-CoV-2 antibodies among blood donors in the cities of Wuhan, Shenzhen, and Shijiazhuang of China. Design: Cross-sectional study Setting: Three blood centers, located in the central, south and north China, respectively, recruiting from January to April 2020. Participants: 38,144 healthy blood donors donated in Wuhan, Shenzhen and Shijiazhuang were enrolled, who were all met the criteria for blood donation during the COVID-19 pandemic in China. Main outcome measures: Specific antibodies against SARS-CoV-2 including total antibody (TAb), IgG antibody against receptor-binding domain of spike protein (IgG-RBD) and nucleoprotein (IgG-N), and IgM. Pseudotype lentivirus-based neutralization test was performed on all TAb-positive samples. In addition, anonymous personal demographic information, including gender, age, ethnicity, occupation and educational level, and blood type were collected. Results: A total of 519 samples from 410 donors were confirmed by neutralization tests. The SARS-CoV-2 seroprevalence among blood donors was 2.29% (407/17,794, 95%CI: 2.08% to 2.52%) in Wuhan, 0.029% (2/6,810, 95%CI: 0.0081% to 0.11%) in Shenzhen, and 0.0074% (1/13,540, 95%CI: 0.0013% to 0.042%) in Shijiazhuang, respectively. The earliest emergence of SARS-CoV-2 seropositivity in blood donors was identified on January 20, 2020 in Wuhan. The weekly prevalence of SARS-CoV-2 antibodies in Wuhan’s blood donors changed dynamically and were 0.08% (95%CI: 0.02% to 0.28%) during January 15 to 22 (before city lockdown), 3.08% (95%CI: 2.67% to 3.55%) during January 23 to April 7 (city quarantine period) and 2.33% (95%CI: 2.06% to 2.63%) during April 8 to 30 (after lockdown easing). Female and older-age were identified to be independent risk factors for SARS-CoV-2 seropositivity among donors in Wuhan. Conclusions: The prevalence of antibodies to SARS-CoV-2 among blood donors in China was low, even in Wuhan city. According to our data, the earliest emergence of SARS-CoV-2 in Wuhan’s donors should not earlier than January, 2020. As most of the population of China remained uninfected during the early wave of COVID-19 pandemic, effective public health measures are still certainly required to block viral spread before a vaccine is widely available.