The invention discloses a gene expression regulation system and application thereof. The gene expression regulation and control system comprises a plasmid composition for gene expression regulation and control, the plasmid composition comprises a plasmid 1 and a plasmid 2, and the plasmid 1 is a plasmid with an expression nucleotide sequence as shown in SEQ ID NO.1; the plasmid 2 is a plasmid for expressing mini-CRISPR, and the mini-CRISPR contains a target sequence and a nucleotide sequence as shown in SEQ ID NO.5 at the 5'end and the 3 'end of the target sequence. The I-F type CRISPR-Cas double-plasmid system provided by the invention can accurately identify and combine a promoter region of a target gene, and effective inhibition on transcription expression of the target gene can be realized through conventional plasmid transformation. The system has obvious simplicity and convenience in bacterial gene function research, and meanwhile has wide application prospects in the aspects of bacterial physiology research, metabolic pathway transformation, natural compound synthesis, drug target discovery, novel drug development, high-throughput screening and the like.