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Development of noninvasive bioimaging fluorescent probes for detecting particular enzyme activity is greatly recommendable for preclinical diagnosis of cancer. Given that the elevated β-gal activity is positively correlated with several tumors, developing a fluorescent probe for the sensing of β-gal is therefore highly desirable for cancer diagnosis. Herein, a new enzyme-activatable near-infrared (NIR) turn-on fluorescent probe (DMC-βgal) was developed for accurately detecting β-gal activity characterized by excellent selectivity, high sensitivity (LOD = 0.298 U/L), and low toxicity. More importantly, DMC-βgal qualifies remarkable NIR excitation (725 nm) and emission wavelength (770 nm), an ideal tool for restrained photodamage and suppressed autofluorescence. The above excellent performance of DMC-β-gal allowed for the accurate monitoring of endogenous β-gal in living cells. Moreover, the probe was successfully applied to detect intracellular β-gal activity in different types of cancer cells, verifying that SKOV-3 cells had a higher level of β-gal activity than those of A549, HCT-116, MCF-7, and HepG2 cells. Furthermore, DMC-βgal could real-time visualize endogenously β-gal in tumor-bearing nude mice with low auto-fluorescence interference. All results fully demonstrated that DMC-βgal has potential value as a promising strategy for diagnosis of β-gal-related diseases. [Display omitted] • A new near-infrared excited and emitted β-galactosidase (β-gal) fluorescent probe DMC-βgal was synthesized. • The probe DMC-βgal is characterized by excellent selectivity and high sensitivity (LOD = 0.298 U/L). • The probe is applied to prove that the β-gal activity in SKOV-3 cells is higher than HepG2, A549, HCT-116, MCF-7 cells. • DMC-βgal can real-time visualize the β-gal activity in tumor-bearing nude mice with low photodamage and autofluorescence. [ABSTRACT FROM AUTHOR]