Abstract: A gene ansB knockout mutant of Citrobacter werkmanii and an application thereof are provided. The gene ansB knockout mutant of the C. werkmanii is C. werkmanii with a gene ansB knocked out and a nucleotide sequence of the gene ansB is as shown in SEQ ID NO: 1. In the present invention, the acquired engineering bacteria with the gene ansB of the C. werkmanii knocked out are cultured in LB, TSB, NB and other media at 25° C. and 30° C., so that a biofilm formation capacity of the C. werkmanii on a polypropylene material is improved. Thus, the application scenarios and scopes of the C. werkmanii in heavy metal ion adsorption and construction of cellular protein synthesis micro-factories are broadened.