The invention discloses a linoleate isomerase mutant and application thereof, and the linoleate isomerase mutant is obtained by mutation of 62th-site methionine of linoleate isomerase with an amino acid sequence as shown in SEQIDNO:1 into alanine or glycine, and mutation of 295th-site serine into alanine, leucine or valine. The enzyme activity for catalysis of linoleic acid for preparation of trans-10,cis-12 conjugated linoleic acid of the linoleate isomerase mutant can be greatly improved compared with that of a wild-type enzyme, especially during double mutation such as M62A / S295L, M62A / S295A, M62A / S295V, M62G / S295L, M62G / S295A and M62G / S295V, the enzyme activity can be increased by about 10 times, and the linoleate isomerase mutant has good prospects for industrial application.