Hydrazine (N2H4) finds use in a wide spectrum of industries. However, it is highly hazardous and leads to the environmental pollution and the subsequent potential health risk. Therefore, the development of new sensing techniques for pollutants is a significant challenge. Herein, we report a fluorescence and colorimetric sensing of N2H4 through a D-π-A structure including benzothiazole and benzothiazolium units as electron donating and withdrawing cores, respectively. The sensing mechanism is based on the ratiometric change of emission intensity resulted from the change in electron density distribution in the presence of N2H4 triggered by selective nucleophilic addition to –C[dbnd]N+–, not reported yet such detection strategy for N2H4. The response is very selective over a series of anions, cations, amines, amino acids. Besides, the low cytotoxic probe is able to visualize the subcellular distribution of N2H4 in HeLa cells by confocal fluorescence microscopy. The sensing process parameters such as N2H4 level, time and pH are modeled by Box-Behnken design with response surface methodology. The practical applications such as a low-cost electronic diagnostic platform, a smartphone sensing system, a cotton swab, an onion tissue imaging, and a soil sample imaging is successfully established.