Senescence is an important biological process, which leads to the gradual degradation of its physiological function and increases morbidity and mortality. Herein, a novel ratiometric fluorescent probe (P1) was constructed by using benzothiazolyl acetonitrile dye as fluorophore, exhibiting significantly enhanced blue-shifted emission to indicate the activity of β-galactosidase (β-gal), a commonly used biomarker for the detection of senescent cells. After incubation with β-gal, the excimer emission of P1 at 620 nm was weakened, while the emission at 533 nm was significantly enhanced, forming an obvious ratiometric probe with high sensitivity and low detection limit (2.7 mU·mL-1). More importantly, probe P1 can locate lysosomes accurately, allowing us to monitor the emergence of living cell senescence in real time. P1 was successfully used to detect β-gal activity in PC-12 cells, Hep G2 cells, and RAW 264.7 cells. It showed strong green fluorescence signal in senescent cells and red fluorescence signal in normal cells, indicating that it can detect endogenous senescence-related β-gal content in living cells. For in vivo drug-induced senescence imaging, after 5 weeks of injection of D-galactose or hydroxyurea, the mice showed significant fluorescence enhancement in specific channels to indicate the activity of β-gal in vivo. At the same time, the senescence of cell-specific organs and skin tissues at the organ level were also detected, which proved that the drug-induced senescence of brain, skin, and muscle tissues was the most serious. These results supported the important application value of P1 in senescence biomedical research.