Precise recognition of senescent cells is essential owing to their key role in various diseases, including aging and tumor suppression. Although senescence-associated β-galactosidase (SA-β-Gal) is widely used as a senescence biomarker, it is not remarkably accurate due to its overexpression in some non-senescent cells. Herein, we developed a dual-channel fluorescent probe to improve the identification accuracy of senescent cells through simultaneous detection of β-gal and α-l-fucosidase (AFU) because the two markers are upregulated in senescent cells. The dual-channel fluorescent probe named HDQ-NA-AFU-Gal was employed to detect β-gal and AFU and identify senescence in living cells and tumor-bearing mice. When the two are present, the dual-enzyme activated probe emits strong red and green fluorescence at 740 nm and 550 nm, respectively, enabling independent detection of β-gal and AFU. This dual-enzyme detection approach allows for the precise differentiation between normal and senescent cells, particularly in ovarian cancer cells overexpressing β-gal. Furthermore, the probe can be applied as an effective tool for tracing β-gal and AFU during tumor senescence in mice. Thus, the dual-enzyme-responsive fluorescent probe has promising applications in biological research and clinical medicine.