Lactosucrose is a kind of trisaccharide that functions as a significant prebiotic in the maintenance of gastrointestinal homeostasis for human. In this study, a levansucrase from Brenneria goodwinii was further used for the lactosucrose production. The recombinant levansucrase showed efficiency in the lactosucrose production by transfructosylation from sucrose and lactose, and no other oligosaccharide or polysaccharide was detected in the reaction mixture. The transfructosylation product by this recombinant enzyme was structurally determined to be lactosucrose by FT-IR and NMR. The production condition was optimized as pH at 6.0, temperature at 35 degrees C, 5UmL(-1) enzyme, 180gL(-1) sucrose, and 180gL(-1) lactose. Under the optimal condition, the enzyme could approximately produce 100gL(-1) lactosucrose when the reaction reached equilibrium. The recombinant levansucrase could effectively and exclusively catalyze the formation of lactosucrose, which might expand the enzymatic choice for further preparation of lactosucrose.