The invention provides a genetic engineering strain being capable of producing acetyl coenzyme A oxidase and having good heat stability as well as a construction method and applications of the genetic engineering strain. The engineering strain provided by the invention has the gene sequence as shown in SEQ ID NO.1. A segment of gene encoded with the acetyl coenzyme A oxidase is cloned from a candida tropicalis strain, and the gene has the sequence as shown in SEQ ID NO.2; the gene is subjected to random mutation by adopting error-prone PCR, the carrier self-constructed is subjected to pre-expression and screening in escherichia coli, and then the acetyl coenzyme A oxidase genetic engineering strain having high yield and good stability is obtained. In the applications of producing acetyl coenzyme A oxidase through the fermentation method, the engineering strain is high in production capacity, the amino acid sequence of the acetyl coenzyme A oxidase is shown as SEQ ID NO.3, and the engineering strain is applied to the large-scale industrial production of acetyl coenzyme A oxidase.