Objective To evaluate the effects of knowledge about malaria after the implementation of Global Fund Malaria Project in Chongqing for future prevention and treatment of malaria. Methods Four counties were selected from the counties of Global Fund Malaria Project of Chongqing. Three towns were randomly selected in each selected county; one primary school and one high school were investigated in each selected town; one class was randomly selected in each selected school; and 30 students in each selected class were randomly selected. Then, three villages were randomly selected in each selected county; one group of villagers were randomly selected in each selected village; and 200 local residents were randomly investigated in each selected villager group. Before and after implementation of Global Fund Malaria Project, their awareness of malaria was investigated using a questionnaire. Results After implementation of the project, the pupils’ average awareness rate of knowledge about malaria rose from 58.94% to 89.96% (χ2=179.48, Plt;0.01) with average increase of 31.02%; the middle school students’ average awareness rate of rose from 52.83% to 86.06% (χ2=196.64, Plt;0.01) with average increase of 33.23%; the local residents’ average awareness rate of knowledge about malaria rose from 56.74% to 83.89% (χ2=1 281.70, Plt;0.01) with average increase of 27.15%. The accuracies of all respondents after project implement was higher than before. After the implementation, the accuracies of malaria transmission route, main clinical symptoms, and seeing doctor while malaria occurring were all above 80%, but the accuracy of the index of best preventive methods was less than 50%. Conclusion The implementation of Global Fund Malaria Project in Chongqing increase the awareness rate of malaria knowledge but the relevant education should be strengthened, so as to promote general population’s awareness of malaria prevention.