The present invention relates to the field of genetic engineering and hereditary engineering. The present invention discloses a β-galactosidase (β-galactoside galactohydrolase, EC 3.2.1.23) mutant with high transglycosidase activity, which is obtained by single-site-saturation mutation of amino acid sequences of β-galactosidase from Aspergillus candidus and Aspergillus oryzae, with own signal peptides removed. The transglycosidase activity of the mutant is over 15% higher than that of wild types. Meanwhile, the present invention also discloses a DNA molecule which encodes the mutant, a recombinant expression vector containing the DNA molecule, and a host cell expressing the DNA molecule. In addition, the present invention also provides a method for preparing β-galactosidase mutant with high transglycosidase activity by using the recombinant expression vector and applications of the mutant, the DNA molecule, the recombinant expression vector and the host cell in preparation of β-galactosidase.