Abstract: Objective To obtain DNA aptamers with a highly specific affinity to HIV gp41 antigen using SELEX screening for detection of HIV. Methods The specific DNA aptamers of HIV gp41 antigen were screened from the double-stranded DNA derived from the single-stranded DNA (ssDNA) library with agarose beads as the supportive medium and HIV gp41 antigen as the target molecule using SELEX technique and real-time quantitative PCR. Results The secondary ssDNA library obtained after 6 rounds of screening was amplified by PCR to obtain dsDNA. The dsDNA was linked with pMDTM 18-T vector, cloned and sequenced to obtain 4 aptamers of HIV gp41 antigen. The affinities of the 4 aptamers (Kd) all reached the nanomolar level. Among the 4 aptamers, the No.15 aptamer showed the strongest affinity. Specificity analysis of the aptamers revealed that all these 4 aptamers had specific affinity to HIV gp41 antigen with no affinity to other non-specific proteins. Conclusion We successfully obtained DNA aptamers with highly specific affinity to the HIV gp41 antigen from random single-stranded oligonucleotide library, and the obtained aptamers have the ability to antagonize HIV gp41 antigen.