Development of fluorescent probes for on-site sensing and long-term tracking of specific biomarkers is particularly desirable for the early detection of diseases. However, available small-molecule probes tend to facilely diffuse across the cell membrane or remain at the activation site but always suffer from the aggregation-caused quenching (ACQ) effect. Here we report an enzyme-activatable aggregation-induced emission (AIE) probe QM-βgal, which is composed of a hydrophilic β-galactosidase (β-gal)-triggered galactose moiety and a hydrophobic AIE-active fluorophore QM-OH. The probe is virtually non-emissive in aqueous media, but when activated by β-gal, specific enzymatic turnover would liberate hydrophobic AIE luminogen (AIEgen) QM-OH, and then highly fluorescent nanoaggregates are in situ generated as a result of the AIE process, allowing for on-site sensing of endogenous β-gal activity in living cells. Notably, taking advantage of the improved intracellular retention of nanoaggregates, we further exemplify QM-βgal for long-term (∼12 h) visualization of β-gal-overexpressing ovarian cancer cells with high fidelity, which is essential for biomedicine and diagnostics. Thus, this enzyme-activatable AIE probe not only is a potent tool for elucidating the roles of β-gal in biological systems, but also offers an enzyme-regulated liberation strategy to exploit multifunctional probes for preclinical applications.