Galactooligosaccharide (GOS) is an oligosaccharide composed of galactose molecules and glucose. It is considered a prebiotic, due to the good effects and specific changes in the composition and activity of gastrointestinal microbiome, once it is a selectively fermented ingredient. GOS can be produced by the β-galactosidase catalyzed conversion of lactose, known as transgalactosylation reaction and occurs in high lactose concentration. The Kefir grains are formed by several β-galactosidase producing microorganisms in symbiosis, being a rich source of probiotics. The objective of this study was to isolate and select β-galactosidase producing microorganisms from kefir that was growing in different substrates, for the consequent synthesis of GOS with the permeabilized cells. The kefir grains were inoculated in the substrates: 1- raw milk; 2- pasteurized milk; 3- cheese whey 40%; 4- lactose 40% and, after isolation, the strains were placed in a lactose-rich medium for 72 hours. Next, the cells were permeabilized using a 50% ethanol solution and then the hydrolytic activity of β-galactosidase was monitored using the oNP-Gal as substrate of the reaction. As a result, three strains stood out in the production of the enzyme: one from substrate 1: J (0.46 U/mL); and two from substrate 4: A (0.67 U/mL) and C (0.46 U/mL). Then, the permeabilized cells were placed in a 20% lactose solution for GOS conversion and compared with commercial β-galactosidase enzyme (Lactozyme®). GOS quantification was performed in HPLC and the strain J (43.28 g/L) produced the highest amount of GOS, even compared with the commercial enzyme (28.18 g/L). Therefore, it can be concluded that microorganisms isolated from kefir have a great potential for β-galactosidase production. In addition, the application of these enzymes to the synthesis of GOS is interesting, since this reaction is highly dependent on the enzyme properties and the production cost could also be lower.