Uropathogenic Escherichia coli (UPEC) is the leading causative agent of Urinary tract infection (UTI). The emerging multidrugresistance of UPEC to antimicrobial agents (such as antibiotics or Chinese herbs) has been a serious public health problem and may result in the complication or failure of UTI therapy, yet with unknown underlying mechanisms. Our present study aims to explore the plasmid DNA-based gene transfer mechanism of the drug resistance UPEC strains. The pPICZαA plasmid containing the Zeocin resistance gene was applied as extraneous resistance plasmid to investigate the uptake ability of plasmid and the plasmid transfer frequency of a UPEC stain alone or co-culturing with DH5α bacteria during the formation of biofilm process. The results showed that the uptake efficiency and plasmid transfer frequency increased from 8 h to 24 h during the biofilms formation, and then slowed down after the formation of biofilms. What’s more, the plasmid transfer frequency was independent of the bacteria culture conditions. Our present study is of importance to the mechanisms illustration of multidrug resistance (like herbal medicine) in UPEC, and might provide a foundation for the research of resistance gene spreading reduction.