The invention relates to a novel method for evolving target protein in vivo by using iteration homologous recombination. The method is characterized by including: preparing single-stranded nucleotide with mutation by using two-step PCR in vitro, digesting the nucleotide by DNaes I, recovering 80-100nt fragment to form a single-stranded nucleotide mutant library of target protein, transforming the single-stranded mutant library into Escherichia coli, establishing Escherichia coli flora containing mutant of target protein by using lambda-Red homologous recombination technology, transforming the single-stranded mutant library into Escherichia coli flora containing mutant of target protein to form Escherichia coli flora with large library capacity, cycling the operation of transforming the single-stranded mutant library into Escherichia coli for 2-6 times to form Escherichia coli mutant library containing target gene mutation, and obtaining the target protein mutant by using highly effective screening or high throughput screening. The technology can be used for evolving microbial gene in situ, and can improve the enzyme catalytic activity, and can be used for improving the yield of microbial metabolite.