The rapid and sensitive detection of pathogenic bacteria is very important for timely prevention and treatment of foodborne disease. Here, a bacteria-imprinted conductive poly(3-thiopheneacetic acid) (BICP) film-based impedimetric sensor was developed for the rapid, sensitive and label-free detection of staphylococcus aureus (S. aureus). The BICP film preparation was very convenient and eco-friendly, which was in situ deposited on gold electrode surface without the use of toxic organic solvents and cross-linkers. The process of imprinting and recognition were characterized by electrochemical technique and scanning electron microscope. The BICP had a novel structure without cocci-shaped cavities formed in the poly(3-thiopheneacetic acid) (PTAA) matrices. To obtain the optimal sensing performance, a set of factors affecting the imprinting and recognition were investigated. Under the optimized conditions, an extremely rapid recognition within 10 min, a very low limit of detection (LOD) of 2 CFU/mL, and wide linear range from 10 to 108 CFU/mL were achieved by the BICP film-based impedimetric sensor. The sensor also demonstrated high selectivity, and good universality and repeatability. Furthermore, the feasibility of its application has also been demonstrated in the analysis of real milk samples. This sensor offered a simple and universal method for rapid, sensitive, and selective detection of pathogenic bacteria, which could hold great potentials in fields like food safety.