The invention provides a biosensor for detecting uracil glycosylase. The biosensor comprises an UDG template, an S-HAP1 hybridization chain, HAP2-AgNCs and ExoIII enzyme, fluorescence detection is employed for detecting the activity of the uracil glycosylase, an excitation wavelength of the fluorescence detection is 560 nm, the emission wavelength is 625 nm, and a detection wave band is 575-750 nm. The biosensor has the advantages of good specificity, high sensitivity, mild reaction condition, and fast reaction speed; silver cluster detection is employed, operation is simple, a detection period is short, and carrying is easy; the technology cost is low, and the technology is suitable for low-cost requirement in industrialization; the preparation method is simple, the performance is stable,the repeatability is good, and the method is suitable for detecting UDG in field of medical treatment and public health.