The invention discloses an oligonucleotide-linker-mediated DNA assembly method and an application thereof. The assembly method comprises steps of synthesis of a long sequence, preparation and assembly of a module carrier, remolding and assembly of a skeleton carrier, preparation of a short sequence, and enzyme-cut and link-up integration of the module carrier, the remolded skeleton carrier and the short sequence. According to the method, the short sequence is a regulating element such as a promoter and an RBS, and the long sequence is a replication initiation point of a gene or a carrier. The short sequence is designed to synthesize a double-stranded DNA chain, and can connect the long sequence and be used as a barcode sequence to determine the assembly sequence in an assembly process. The gene sequence in an operon is achieved by synthesizing short sequence with different cohesive ends. According to the method, multiple factors are allowed to participate in gene regulation at the same time. The method can be used to construct a combinatorial library of a biosynthesis channel, and a high-flux optimized channel is achieved.