The reported positive rates of hepatitis B virus surface antigen (HBsAg) among Chinese blood donors generally do not include data from pre-donation rapid tests. The aim of this study was to estimate the prevalence of serologic markers for HBsAg and risk factors among blood donors from Chengdu, China. From April 2010 to March 2011, a total of 16,875 blood donors were enrolled. Data from HBsAg tests before and after donations were collected to estimate the prevalence of HBsAg. A case-control study was conducted in 265 unique HBsAg-positive blood donors and 530 seronegative donors between January 2011 and October 2012. The odds ratio (OR) and 95% confidence interval (CI) were calculated using conditional logistic regression. The population attributable risk (PAR) was estimated. The prevalence of HBsAg was 3.17% (95%CI, 2.91-3.43%). The following risk factors were associated with HBV infection: non-vaccination for HBV (OR = 4.236; 95%CI, 2.72-6.60%), razor sharing (OR = 2.370; 95%CI, 1.46-3.84%), dental treatment (OR = 1.714; 95%CI, 1.21-2.43%), acupuncture (OR = 1.983; 95%CI, 1.20-3.28%), a family history of HBV infection (OR = 2.257; 95%CI, 1.32-3.86%), and endoscopy (OR = 2.17; 95%CI, 1.04-4.51%). The PAR values of the risk factors were 42.77%, 31.78%, 13.68%, 6.18%, 6.09%, and 2.85%, respectively, and the total PAR was 68.78%. The prevalence of HBsAg among Chinese blood donors is still high. HBV vaccinations can provide protection. Blood contact from sharing instruments is still a high risk route of transmission for HBV. Immunization programs and behavioral interventions should be used to prevent blood donation infections and improve blood safety.