Numerous excellent fluorogenic probes for NO detection based on NO-mediated reactions have been developed. However, some of them still suffer from limitations such as low selectivity, slow response, and a short excitation wavelength (<500 nm). Herein, a novel two-photon fluorogenic probe (XNO1) based on a Schiff base derivative has been reported for the first time. This new mechanism with a Schiff base structure as the specific response moiety towards NO endows the probe with fast responsibility, high selectivity and pH-independent properties. Furthermore, XNO1 has been demonstrated to be lysosome-targeted and to successfully monitor exogenous/endogenous NO in living cells and zebrafishes with one- and two-photon fluorescence imaging.