The invention provides a method for parallel determination of the activity of uracil-DNA glycosylase and endonuclease IV, application thereof and a reagent kit. When a target object is UDG, if UDG exists, U basic groups in a hairpin probe are removed, an AP locus is generated, the generated AP locus is cut through tool enzyme Endo IV, and a primer sequence containing free 3' terminal is released and used for initiating subsequent rolling circle amplification reaction (RCA); if UDG does not exist, the 3' terminals are closed in the hairpin probe, the RCA process can not be carried out. When a target object is Endo IV, if Endo IV exists, the RCA process is the same as that in UDG activity detection; if Endo IV does not exist, the 3' terminals are closed in the hairpin probe, and the RCA process can not be conducted. The hairpin probe can be used as a recognition probe of UDG and can also be used for precursor recognition probe of Endo IV, and accordingly design of the probe is simplified. The detection limits of UDG and Endo IV are reduced to 0.00017 U/mL and 0.11 U/mL respectively, and results are better than or the same as those of other label-free fluorescence methods.