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• An ultra-sensitive fluorescent probe TC-gal with high affinity to β-galactosidase was developed. • The detection of β-galactosidase with low background fluorescence and high signal-to-noise ratio was realized by the response of "turn-on" fluorescence of probe TC-gal. • The visualization of endogenous β-galactosidase in ovarian cancer cells and senescent cells was achieved. • The sensing mechanism based on a competition of TICT with ICT provided a novel idea for design of fluorescent biosensor. The detection of β-galactosidase is significant for identifying the occurrence of ovarian cancer and cellular senescence. Since the complexity and diversity of biological systems, fluorescent probes for biological detection are required to own good selectivity, high selectivity and strong anti-interference ability. To meet this challenge, we designed a "turn-on" fluorescent probe TC-gal by adjusting the effect of twisted intramolecular charge transfer (TICT) between intramolecular charge transfer (ICT) of the fluorophore for ultra-sensitive detecting of β-galactosidase. The β-galactosidase can specifically trigger an enhanced (more than 800 folds) light-up fluorescence of TC-gal due to attenuated TICT effect of tetraphenylethylene part and increased ICT effect induced by electron donating effect of generated hydroxyl. Such "turn-on" fluorescence selectively triggered by β-galactosidase endows probe TC-gal with low background fluorescence and high signal-to-noise ratio. It is demonstrated that designed TC-gal exhibits high selectivity, ultra-high sensitivity (LOD = 8.4 × 10−5 U/mL) and strong affinity (Km = 18.4 μM) to β-galactosidase in buffer solution with good anti-interference ability to other biological analytes. Furthermore, biocompatible probe TC-gal is successfully applied to detect endogenous β-galactosidase in ovarian cancer cells and distinguish senescent cells. We anticipate this work can inspire the generation of superior fluorescent biosensors for biological and medical application. [ABSTRACT FROM AUTHOR]