β-Galactosidase is a key enzyme in the preparation of galacto-oligosaccharides. In this paper， the β-galactosidase gene lacZ of wild strain Klebsiella B5582Y was harvested and used to successfully construct the recombinant plasmid pET28a-24 of E. coli Induced by IPTG， the activity of recombinase increased nearly 10 times than before. The best process conditions for the synthesis of galacto-oligosaccharide by purified recombinant enzymes through single factor and response surface method are as follows： added enzyme amount 130 U/mL， temperature 44 ℃， substrate concentration 90%， pH value 7.0-7.5 with a reaction time of 10 h， the yield of galacto-oligosaccharides reached 44%. Recombinase shows good transglycoside activity and has great application potential in the field of galacto-oligosaccharide synthesis.