Reactivity based fluorescent probes have been widely investigated as a powerful and noninvasive tool for disease diagnosis in recent years. β-Galactosidase (β-gal), one of the typical lysosomal glycosidases, is reported to be a vital biomarker overexpressed in primary ovarian cancer cells. Fluorescent probes with excellent performance for endogenous β-gal detection offer a unique option for visualization and diagnosis of primary ovarian cancer cells. Herein, a near-infrared fluorescent probe Lyso-Gal with lysosome-targeting ability was developed for lysosomal β-gal detection and imaging in ovarian cancer cells (SKOV-3 cells). Lyso-Gal exhibits weak fluorescence in aqueous solution but emits bright NIR fluorescence at 725 nm after incubation with β-gal. Highly selective imaging of ovarian cancer cells has been achieved upon incubation with Lyso-Gal for only 1 min. The detection time is extremely short. In comparison with a similar hemicyanine probe, Hx-Gal, without lysosome-targeting ability, Lyso-Gal realizes endogenous β-gal visualization in lysosomes and shows brighter fluorescence than Hx-Gal in SKOV-3 cells. This work demonstrates the potential of Lyso-Gal for detection of primary ovarian cancer cells by using β-gal as the biomarker.