β-Galactosidase (β-gal) which is overexpressed in primary ovarian cancer can be employed as a valuable biomarker for ovarian cancer. Thus, monitoring and imaging endogenous β-gal in living cells is of great importance. Herein, a dicyanoisophorone-based near-infrared (NIR) fluorescent probe 2-(5,5-dimethyl-3-((E)-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)styryl)cyclohex-2-en-1-ylidene)malononitrile named DP-βgal, was rationally designed and synthesized for the monitoring of β-gal activity in living cells. In the presence of β-gal, with the breaking of the glycosidic bond, the NIR fluorescence of the dicyanoisophorone derivative gradually recovered, enabling the fluorescence “off-on” quantitative determination of β-gal activity. DP-βgal has the advantages of good selectivity and high sensitivity for the detection of β-gal, with the limit of detection (LOD) of 3.2 × 10−3 U. Furthermore, based on its advantages of long-wavelength emission and excellent biocompatibility, the practical applications of DP-βgal in NIR imaging of β-gal in living ovarian cancer cells (SKOV-3) were demonstrated. Display omitted •An “off-on” NIR fluorescent probe (DP-βgal) was rationally constructed for the detection of β-galactosidase (β-gal).•DP-βgal shows high sensitivity, compatibility and selectivity toward β-gal with large Stokes shift and NIR emission.•A method for diagnosis by imaging of endogenous β-gal in living ovarian cancer cells was developed.