Both developing and developed countries are facing difficulties in finding regular donors. In areas that are exposed to frequent conflicts and wars, such as the Gaza Strip, there is a need for a continuous blood supply. This study aims to determine the level of knowledge, attitudes, and practices toward blood donation in the Gaza Strip, Palestine. A cross-sectional study was conducted in 2022, in which 1506 participants were randomly selected from different governorates in the Gaza Strip. A structured and valid questionnaire was employed to assess the level of knowledge, attitudes, and practices toward blood donation. All statistical analyses were performed using SPSS version 28. The chi-square test was used to measure the significance of associations. A total of 1506 individuals living in the Gaza Strip participated. The total mean score of the overall knowledge and positive attitudes toward blood donation was 55.1% and 67.1%, respectively. Furthermore, 1236 (82.1%) of the study participants never donated blood. Of them, 260 (21.0%) demonstrated that they do not have information on when, where, and how to donate; 228 (18.4%) thought that they were not fit to donate; 187 (15.1%) demonstrated that they did not have time to donate; 143 (11.6%) feared health problems, and 132 (10.7%) feared anemia. On the contrary, 99 (36.7%) donated blood when a friend or relative needed blood, and 171 (63.3%) were voluntary donations. Statistically, a significant association was found between knowledge, attitudes, practices, and sociodemographic variables (p < 0.05 for all). The study findings indicated poor donation habits despite positive attitudes toward blood donation in the Gaza Strip, Palestine. This research emphasizes the need to recognize and correct the knowledge gap that results in unfavorable behaviors against blood donation.