Objective: To assess the knowledge, attitudes and practices regarding blood donation in the general population. Methods: A cross-sectional study was conducted and enrolled 416 people between 18 and 65 years old at random, living in three health zones in the city of Bukavu in eastern DRC. These subjects responded to a questionnaire on knowledge, attitudes and practices regarding blood donation. Results: Our sample consisted of 61.5% men, 70.9% of people from one level of education at the secondary level and 60% unemployed. According to the surveyed subjects blood was considered as a fuel of the body in 44.6% and as a source of life in 44.1%. Sixty-one percent of the population did not know the practice of blood donation; this knowledge differed significantly (p < 0.0001) according to the level of education. 67.1% of people knew that the blood had negative effects and 27.4% did not know where the blood bags were stored. Channels of knowledge about blood donation practices were associations of blood donors (30.9%), awareness campaigns (18.2%), school (17.3%) and media (15.5%), churches (10%), and friends (8.1%). Approximately 85% of subjects who knew blood donation agreed with blood donation and blood transfusion but only 54.9% had donated blood in their lifetime, with a proportion of loyalty to blood donation (31.8%). In the population 59.4% were willing to donate blood. Motivation to donate blood most common (66%) was volunteerism while 19.1% were willing to donate blood for a J. M. Kabinda et al. 2526 family member and 12.6% willing to donate blood against money. The reasons for refusal to donate blood identified were fear of contracting diseases especially HIV, lack of information, religious beliefs, the sale of the collected blood by medical staff, and fear of test result for HIV. Conclusion: Factors of refusal to donate blood were identified. Efforts and new strategies tailored to these factors must be developed and implemented to increase blood donation.