Mass and signal transfer, dispersion of reactive metabolites in living cells, and interactions between biomacromolecules are greatly affected by viscosity inside the cells. It is crucial to accurately determine viscosity for reliable results because of the complexities of live cells. Herein, we introduce a new fluorescence probe based on the cyanobiphenyl and benzothiazolium units. This probe not only responds to intracellular viscosity but also detects hydrazine, a widely used chemical that poses significant environmental and toxic risks to organisms. The proposed sensing mechanism provides a new pathway that includes intramolecular cyclization with hydrazine, which differs from other sensing mechanisms. A weak emission (at 590 nm) of the probe under excitation at 365 nm resulted in 25-fold higher emission at 488 nm after the addition of N2H4. The quantum yield of the probe (Φ = 0.089) increased to Φ = 0.199 with the addition of N2H4. In addition, the probe demonstrated 45-fold emission enhancement at 560 nm in viscous media, with a color change from non-fluorescence to yellow fluorescence. Good hydrazine sensing features with high adaptability, selectivity, sensitivity, ratiometric and fast response (90 s), low cytotoxicity (more than 90% of cell viability), low detection limit (86.0 nM), good linearity in the range of 0–35.0 μM, and high signal-to-noise ratio sensing capability were achieved. The hydrazine-sensing capability of the mitochondria-targetable probe in living cells makes it a strong candidate for various biological and environmental applications, including intracellular tracking and imaging. These results suggest that the present probe shows significant potential for the effective fluorescence detection of hydrazine.