Endonuclease plays a vital role in a variety of biological processes and the assay of endonuclease activity and inhibitors is of high importance in the fields ranging from biotechnology to pharmacology. Howerer, traditional techniques usually suffer from time intensive, laborious, and cost-expensive. This work aims to develop a facile and sensitive method for endonuclease activity assay by making use of the fluorescence enhancement effect when DNA-silver nanoclusters (DNA-Ag NCs) are in proximity to guanine-rich DNA sequences. The system mainly consists of block DNA (B-DNA), G-DNA and Ag-DNA. B-DNA serves as the substrate of the endonuclease (S1 nuclease as the model enzyme). G-DNA, which is predesigned entirely complementary to B strand, contains a guanine-rich overhang sequence and hybridization part at the 5'-end. Ag-DNA involves a sequence for Ag NCs synthesis and a sequence complementary to the hybridization part of the G-DNA. In the "off" state, B-DNA plays the role as a blocker that inhibit the proximity between Ag NCs and guanine-rich DNA sequences, resulting in a low fluorescence readout. However, if S1 nuclease is introduced into the system, B-DNA was cleaved into mono- or short-oligonucleotides fragments, which could not hybridize with G-DNA. As a result, the subsequent addition of DNA-Ag NCs could bring guanine-rich DNA sequences close to the Ag NCs, accompanied by a significant fluorescence enhancement. Therefore, endonuclease activity could be successfully quantified by monitoring the variation in fluorescence intensity. In addition, this approach can also be applied for inhibitor screening of endonuclease. This label-free and turn-on fluorescent assays employing the mechanism proposed here for the detection of nuclease and inhibitors turn out to be sensitive, selective, and convenient.