The invention provides a heat-resistant beta-galactosidase mutant with transglycosylation. The mutant is obtained through the steps that beta-galactosidase (BgaB) catalyzes an amino acid site E303 to be mutated into cysteine (Cys) in a site-directed mode from original glutamic acid (Glu), and a cysteine sulfhydryl group is oxidized into -SOO<->. The invention further designs a preparation method and application of the mutant. Beta-galactosidase (BgaB) derived from bacillus stearothermophilus serves as an object, a method combining a site-directed mutant with chemical modification is adopted, the functional evolution efficiency of transglycosylation is significantly improved, the synthesis quantity of galactooligosaccharide is improved, the synthesis quantity of galactooligosaccharide is improved to 11.5% from 0% of wild-type enzyme under a medium temperature and neutral environment, limitation of reacting under an acid environment in the prior art is broken through, and meanwhile hydrolysis of a substrate or a product is avoided when galactooligosaccharide is synthesized.