Oligosaccharides especially prebiotics take high attention in the development of foods because of their physiological properties in human health. They are generally synthetized enzymatically via transferases or hydrolases from mold or bacteria. The fact is that such oligosaccharides synthetized by probiotic bacteria, should be utilized by these microorganisms. This study focused on the production of oligosaccharides with prebiotic potential by crude enzyme preparation from bifidobacteria. Both monosubstrates and bisubstrates systems together with TLC and HPLC techniques, were applied. The crude enzyme preparation has different hydrolase activities such as α-glucosidase (2U/mL), β-glucosidase (0.3 U/mL), α-galactosidase (1.2 U/mL), β-galactosidase (0.4 U/mL), β-fructosidase (11.5 U/mL). Additionally, it also has transglycosylation activities on lactose, lactulose, maltose and sucrose substrates. Two or three types of oligosaccharides were detected. The glycosyltransferase activity peaked at 45 °C, pH 6.6 and 30 g/100 mL substrate concentration. Significant high amount of oligosaccharides were formed in the case of lactose:sucrose combination than others. Both glucooligosaccharides and galactooligosaccharides are detected in the reaction mixtures of bisubstrate. When the lactose is present, the galactosyltransferation is predominated. One-one new types of oligosaccharides were detected in the reaction mixture of bioconversion. Among newly synthetized oligosaccharides, the fraction namely OS4 was utilized by probiotic bifidobacteria only. In conclusion, new types of galacto- and glucooligosaccharides with high prebiotic potentials were synthetized by the crude enzyme from probiotic Bifidobacterium strains.