β-Galactosidase (?β-Gal) is a glycoside hydrolase in cellular lysosomes. It plays a crucial role in hydrolyzing glycoside bonds and even maintaining normal life activities. The development of methods that can accurately and quickly identify β-Gal activity is indispensable for the early diagnosis of ovarian cancer and cellular senescence-causing diseases and the intensive research of molecular biology. Considering the good sensitivity, rapid response, and high temporal and spatial resolution of fluorescent probes, this review summarizes the research progresses on β-Gal fluorescence probes reported in recent years and their effects of disease diagnosis, the molecular design strategies and application characteristics of β-Gal fluorescence probes based on ICT, FRET, AIE, and PET fluorescence-signal transduction mechanisms, and highlights the two-dimensional detection system can break through the limitation of single signal transduction mechanism for probe detection. In summary, rational application of fluorescence mechanism for constructing β-Gal fluorescent probes with high sensitivity, spatial resolution, good chemical stability and strong cell accumulation capacity can advance the highly efficient applications of β-Gal fluorescent probes in biomedical fields, such as diagnosis and therapeutic evaluation of ovarian cancer, cellular senescence assessment, and individual life-span prediction.