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【Objective】 To analyze the HIV infection among voluntary blood donors in Wuhu area, so as to provide effective data for improving blood donation recruitment strategies and clinical safe blood use. 【Methods】 All reactive samples from HIV screening among voluntary blood donors in Wuhu from January 2017 to September 2020 were selected and sent to Wuhu CDC for confirmation test.The receiver operating characteristic (ROC) curve was used to compare the accuracy of enzyme-immune reagents. The optimal critical value of ELISA was used to analyze the setting of serological gray area. The preliminary screening S/CO values of confirmed positive, negative and indeterminate samples were compared, and the significance of differences were analyzed by SPSS 22.0 software. 【Results】 75 cases of initially HIV reactive samples were submitted for testing: 17 cases were confirmed positive, 17 indeterminate and 41 negative.The area under curve (AUC) of the 4th generation anti-HIV ELISA reagent was greater than that of the 3rd generation reagent, and the 4th generation reagent was better than the 3rd generation reagent in accuracy. All the 17 confirmed positive samples had high S/CO reactivity with duplicate reagents, and the initial screening S/CO value was significantly higher than that of confirmed negative and indeterminate samples (P<0.05). There was no significant statistical difference in S/CO values between confirmed negative and indeterminate results (P>0.05). 【Conclusion】 Voluntary blood donors in Wuhu is a middle-endemic population for HIV infection. The setting of grey areas for serological testing is of little significance. Blood safety should be further guaranteed through standardized pre-donation interventions, sensitive nucleic acid detection technologies and shared regional data platforms.