β-galactosidase is an enzyme responsible for catalyzing the hydrolysis of the lactose β-1,4 linkage into α-D-glucose and β-D-galactose. β-galactosidase is found in plants, animals and microorganisms. In Beta-Galactosidase: Properties, Structure and Functions, the authors discuss the main microorganisms that produce β-galactosidase, the characteristics of the culture media, bioprocessing parameters, the most relevant downstream steps used in the recovery of microbial β-galactosidase, as well as the main immobilisation techniques. Next, this compilation examines β-galactosidases classification, mechanisms, characterization and applications. Recent advances in galacto-oligosaccharides production and purification are also covered, and different challenges and future perspectives are discussed. In the closing study, strains with β-Galactosidase activity were isolated from 25 Yak Yoghourt samples collected from the Gannan pasturing area of Gansu Province. An efficient β-galactosidase producing strain SYA2 screened out from 21 strains was identified as Enterobacter sp. by means of morphological feature observation, physiological and biochemical characteristics measurement, and 16S rDNA sequence analysis.