The invention discloses immobilized maleic acid cis-trans isomerase and a preparation method and application thereof, and belongs to the technical fields of enzymology and enzyme engineering and the field of biochemical industries. According to the immobilized maleic acid cis-trans isomerase and the preparation method and application thereof, R5 peptide fragments are connected to the N ends of the maleic acid cis-trans isomerase, R5-MaiA is crosslinked through glutaraldehyde, embedding is conducted on the crosslinked R5-MaiA by utilizing silicon reaction liquid, and immobilization of the maleic acid cis-trans isomerase is completed. The immobilized maleic acid cis-trans isomerase (R5-MI-CLEA-Si) with high stability is obtained through the method which is low in cost and easy to operate, the optimum reaction temperature of the immobilized isomerase is 55 DEG C, and the optimum reaction pH is 7.7; on the condition that the temperature is 55 DEG C, the half-life period of the immobilized isomerase is 4 h, the half-life period of the free isomerase is 0.5 h, the stability of the immobilized isomerase is eight times of that of the free isomerase, and after a catalytic reaction is repeated for eight times, the enzyme activity of the immobilized isomerase can be still retained at 75%.