Abstract: The present disclosure provides a method of preparing a biofilm, comprising: inoculating an source bacteria sample to a substrate by directly dripping the source bacteria sample on the substrate, and culturing the source bacteria sample in a non-cyclic culture medium flow to form the biofilm sample on the substrate. The present disclosure also provides a device for the formation of a biofilm and uses of the biofilm in drug testing and screening. The device and method of the present disclosure saves culture time, reduces contamination, and can be used to form biofilm without anaerobic environment or pH adjustment in culture medium.