Fluorescence imaging-guided diagnostics is one of the most promising approaches for facile detection of tumors owing to its simple operation and non-invasiveness. As a crucial biomarker for primary ovarian cancers, β-galactosidase (β-gal) has been demonstrated to be the significant molecular target for visualization of ovarian tumors. Herein, a membrane-permeable fluorescent chemosensor (namely, LAN-βgal) was synthesized for β-gal-specific detection using the d-galactose residue as a specific recognition unit and LAN-OH (Φ = 0.47) as a fluorophore. After β-gal was digested, the fluorescence of the initially quenched LAN-βgal (Φ < 0.001) was enhanced by up to more than 2000-fold, which exceeded the fluorescence enhancement of other previously reported probes. We also demonstrated that the chemosensor LAN-βgal could visualize endogenous β-gal and distinguish ovarian cancer cells from normal ovarian cells. Further, the chemosensor LAN-βgal was successfully applied to visualize the back tumor-bearing mouse model and peritoneal metastatic ovarian cancer model . More importantly, through spraying, the proposed chemosensor was successfully employed to assist in the surgical resection of ovarian cancer tumors due to its high tumor-to-normal (T/N) tissue fluorescence ratio of 218. To the best of our knowledge, this is the highest T/N tissue fluorescence ratio ever reported. We believe that the LAN-βgal chemosensor can be utilized as a new tool for the clinical diagnosis and treatment of ovarian cancer.