We herein devise a simple and label-free strategy to determine S1 nuclease activity by exploiting the target-induced inhibition of exponential strand displacement amplification (eSDA). In principle, a DNA probe that is designed to produce a large amount of duplexes through a process of eSDA, is degraded by the catalytic activity of S1 nuclease. This reaction blocks the initiation of eSDA, leading to the quitereduced fluorescence of a double-stranded DNA specific fluorescent dye, SYBR Green I compared to the one in the absence of S1 nuclease. With this simple but novel approach, the S1 nuclease activity was selectively assayed with the high sensitivity. In addition, this system was successfully demonstrated to possess the capability to screen potential inhibitors against S1 nuclease.