In this research, food (cheese, yoghurt) and animal (chicken) origin 39 Lactobacillus spp. and human origin (newborn faeces) three Bifidobacterium spp. were used. To designate the β-glycosidase enzyme and specific activities of the cultures, p-nitrophenyl-β-D glikopiranozit (p-NPG) was used as a substrate. The best specific activities between Lactobacilli cultures were observed at Lactobacillus rhamnosus BAZ78 (4.500 U/mg), L. rhamnosus SMP6-5 (2.670 U/mg), L. casei LB65 (3.000 U/mg) and L. casei LE4 (2.000 U/mg) strains. Bifidobacterium breve A28 (2.670 U/mg) and B. longum BASO15 (2.330 U/mg) strains belonging to the Bifidobacterium cultures had the highest specific activity capabilities. Optimization studies were performed to designate the impact of different pH, temperature, and carbon sources on the β-glucosidase enzyme of L. rhamnosus BAZ78 strain (β-Glu-BAZ78), which exhibits high specific activity. As optimum conditions, pH was detected as 7.5, the temperature as 30° C, and the carbon source as 2% glucose for the enzyme. Although the enzyme activity changed as the physiological conditions changed, the β-Glu-BAZ78 showed the highest specificity in the control groups.