Endonuclease V (EndoV) plays important roles in DNA repair. In the absence of a quantitative assay method for EndoV, we have developed a dual enzymatic amplified strategy for the detection of EndoV activity based on a nicking enzyme and a template independent polymerase. Every hydrolysis process performed on a substrate by EndoV can generate only one 3′-hydroxyl terminal to support the polymerization of polymerase, however, with the assistance of a nicking enzyme, abundant 3′-hydroxyl terminals are generated. Next, terminal deoxynucleotidyl transferase (TdT) involved in the second amplified procedure prolongs the 3′-hydroxyl terminus DNA with repeated T bases, providing a long template for the synthesis of fluorescent CuNPs. Consequently, a wide linear dynamic range of 0.02 to 10 U mL⁻¹ is achieved with a detection limit of 0.02 U mL⁻¹. This method exhibits several advantages such as high sensitivity and desirable selectivity, which shows great potential as a promising platform for the sensitive analysis of EndoV or other biomolecules.