Hydrazine (N2H4), as a highly reducing agent and reactive base, is extensively utilized in chemical, pharmaceutical and agriculture fields. However, the high toxicity of N2H4 poses significant risks to human health and can lead to severe ecological contamination. Consequently, it is of significant to develop a tool for the detection of N2H4 in the environment fields and in vivo. Herein, three 1,8-naphthalimide-triphenylamine-based red-emitting fluorescence probes (TSY-1, TSY-2 and TSY-3) have been developed for the detection of N2H4 in actual water sample and in vivo by coupling of 1,8-naphthalimide derivatives with the electron-withdrawing groups (1,3-indandione, malononitrile, and 2-benzothiazolethonitrile) through a N2H4 responsive CC bond. Significant fluorescence quenching of three probes was observed on account of the responses toward N2H4. The detection limits of TSY-1, TSY-2 and TSY-3 for N2H4 were calculated to be 0.2 μM, 0.4 μM and 54 μM, respectively. Utilizations of probe-based test paper strips to qualitatively detect N2H4 in actual water sample by “naked-eye” colorimetry were demonstrated. Additionally, quantitative determination of N2H4 in actual water samples was successfully achieved by the smartphone-based standard curves. Additionally, the applications of TSY-1, TSY-2 and TSY-3 in fluorescence imaging N2H4in vivo are also demonstrated.