A novel turn-on fluorescent chemosensor 4 was designed to distinguish highly toxic hydrazine (N2H4) that relied on a new reaction mechanism. The probe 4 was synthesized from nopinone, which is one of the natural product derivatives. As expected, the developed probe 4 has high selectivity towards hydrazine over various metal ions, anions, amino acids, and other amines. The fluorescence intensity has a positive correlation with the N2H4 concentration from 0 to 900 mu M and the limitation of detection was calculated to be 1.03 mu M. The possible mechanism of probe 4 detecting hydrazine was confirmed by high-performance liquid chromatography (HPLC), high-resolution mass spectrometry (HRMS), and density functional theory (DFT) analysis. Moreover, the cytotoxicity and cell imaging experiments proved that probe 4 has the ability to distinguish hydrazine in living cells.