Uracil-DNA glycosylase (UDG) plays a key role in the base repair system, and detecting its enzymatic activity is crucial for early disease diagnosis. A novel nicking enzyme-assisted, high-specificity fluorescent sensor for uracil-DNA glycosylase activity detection is constructed. A DNA probe modified with uracil was utilized to ligate two free DNA strands to form a newly generated DNA strand. This triggers a nicking enzyme-assisted strand displacement amplification reaction, resulting in the production of single-stranded DNA (ssDNA). Then, the amplified ssDNA triggered the molecular beacons to emit fluorescence. However, the addition of UDG results in the removal of uracil from the DNA probe strand, leaving abasic site (AP site). After heat denaturation, this site was destroyed, preventing subsequent ligation or amplification reactions, resulting in the absence of fluorescence. The findings of our study indicate that the addition of UDG at concentrations exceeding 0.5 U/mL resulted in complete suppression of fluorescence intensity, reaching a value of 0. Conversely, the introduction of elevated levels of other enzymes and proteins, including HAAG, EndoIV, NheI and BSA, did not impede the fluorescence intensity of the system, which remained at 100%. This study presents a specific method for assessing UDG activity that could be valuable for early disease diagnosis in the future.