The invention discloses a construction method and application of a promoter library. The construction method includes the following steps that firstly, an oligonucleotide sequence with promoter features is designed according to a conserved region, a semi-conserved region, a random sequence and a transcription initiation locus of a prokaryote promoter; secondly, the oligonucleotide sequence designed in the first step is subjected to annealing and extension to form double strands, the double strands are connected to a carrier with a report gene, and a mutation library of the promoter is obtained; thirdly, the mutation library, obtained in the second step, of the promoter is migrated into a host cell, a flat plate with corresponding resistance is coated with the mutation library, and the promoter library is obtained. By the adoption of the oligonucleotide sequence which is artificially designed and has the promoter features and the technologies of molecule aggregation and extension, the promoter library with ideal promoter intensity span is obtained. The method is quick, efficient, cheap and small in screening workload.