The disclosure discloses preparation of an L-amino acid deaminase mutant and application thereof, belonging to the technical field of gene engineering. Through pmirLAAD protein modification, analysis of a flexible loop structure around a binding site of the pmirLAAD product, and design of the best mutant, the modification method of the disclosure overcomes the defect that the catalytic efficiency of the previous wild-type enzyme is reduced due to product inhibition, and is tested by experiments. Compared with the control, the catalytic efficiency (1.61 mM−1·min−1) and the product inhibition constant (5.4 mM) of the finally obtained best mutant pmirLAADM4 are respectively increased by 5.2 times and 5.7 times. The yield of α-ketoisovaleric acid can reach 96.5 g/L, and the transformation rate is greater than 97%. By adopting the method of the disclosure, the cost can be greatly reduced, and the industrialization process of production of α-ketoisovaleric acid by an enzymatic transformation method is accelerated.