Abstract: The disclosure discloses an in-vivo continuous directed evolution system and application thereof, and belongs to the fields of gene engineering and enzyme engineering. The system includes Escherichia coli host bacteria carrying a random mutation module mutagenesis plasmid, a programmed death module toxin-antitoxin system and a target gene expression module target plasmid. The modules are coupled with one another, and target genes are subjected to multiple rounds of continuous mutation by virtue of the random mutation module mutagenesis plasmid in the system, so that the mutation rate of the target genes is further increased, and ultimately, efficient evolution and screening of the target genes in the host bacteria are realized. According to the system, mutations are accurately positioned on the target genes, random mutations in non-target gene regions are reduced, and the system has good practical value and can be applied to directed evolution of various different functional proteins.