Base excision repair enzymes played an important role in DNA damage repair, and their detection was related to cancer and disease diagnosis. The traditional detection of base excision repair enzymes was complex and low- sensitive, which only quantitatively analyzed the concentration of enzymes, rather than enzyme activity detection. A signal amplification platform based on catalytic hairpin self-assembly was established to detect endonuclease IV activity in base excision repair enzymes. This method was based on the activation of endonuclease IV acting on the substrate probe and releasing the initiation sequence, which caused the amplification of self-assembly signal of catalytic hairpin to realize the detection and analysis of endonuclease IV activity. According to the fluorescence detection experiment, the detection lower limit of this method was 3.7×10 -7 U/mL, and Endo IV activity could be selectively detected. Therefore, this method was simple in design and operation and high in sensitivity.