Aim: Production of lactulose and other oligosaccharides by Lactobacillus acidophilus NRRL 4495 β-galactosidase and their biological activity. Methodology and Results: The transgalactosylation activity of Lactobacillus acidophilus NRRL 4495 β-galactosidase was investigated under different conditions for synthesis of lactulose and oligosaccharides. The synthesis was optimized with respect to pH; time; enzyme concentration and substrates ratio (lactose: fructose). Maximum production for lactulose was found to be 25 g/L at pH 6.6 with 40: 20% (w/v) lactose to fructose, respectively and enzyme concentration 4 IU/mL after 7 h. With respect to the other oligosaccharides the maximum yield (19 .68 g/L) was obtained under the same conditions but with enzyme concentration 2 IU/mL and after 10 h. As a new pharmaceutical application the produced lactulose and oligosaccharide and their sulfated derivative were found to have fibrinolytic activity, but they failed to act as anticoagulant. Conclusion significance and impact of study: the research leads to increase the production of lactulose and other oligosaccharides with a significant yield and discovered a new pharmaceutical application for all the products.