The invention discloses construction and application of a CRISPR/Cas9 gene editing vector for microorganisms. The constructed CRISPR/Cas9 gene vector consists of a replication start site, a selection marker gene, a Cas9 protein gene, gRNA coding DNA, a homologous recombinant element and an operon. The CRISPR/Cas9 gene vector constructed by the invention is capable of editing (including performing such operations as knocking out, replacing, interpolating and the like on gene or DNA sequence) escherichia coli or corynebacterium glutamicum genome; and the gene vector has the advantages of being short in test cycle, time-saving and cost-saving, high in efficiency and the like.