Abstact: Despite the development of advanced antibacterial biomedical materials, bacterial infection is still a serious problem for indwelling catheter because it usually induces severe complications. Hence, medical indwelling catheter with the capabilities of antibacterial activity, stability, biocompatibility is urgently needed. In this work, a water-insoluble antibacterial coating based on ε-Poly-?-lysine (PL) was prepared via a facile electrostatic interaction between cation PL and anion surfactant, 1,4-bis(2-ethylhexyl) sodium sulfosuccinate (AOT). The ease and efficacy of the PL-AOT complex preparation render it as antibacterial coating applicable to a variety of medical devices for reduction of bacterial infections. This coating was fabricated on the medical catheter with broad-spectrum antibacterial activity, long-term stability, biocompatibility. The contact-killing oriented strategy for the antibacterial action of this coating was confirmed by high-performance liquid chromatography (HPLC). Almost 100% S. aureus and E. coli as Gram-positive and Gram-negative bacteria model could be killed rapidly by this coating for thermoplastic polyurethane (TPU) film. The antibacterial properties of the coated catheters were also assessed under static and dynamic flow conditions. Regardless of the above conditions, the coated catheters displayed remarkable antibacterial activity compared to the uncoated catheters. In addition, this coating showed better antibacterial stability by mimicking the in vivo environment, that is, the antibacterial efficacy could still maintain even after 31?days immersing. Moreover, the coated catheter exhibited negligible cytotoxicity against L929 murine fibroblasts cells. For in vivo experiment, the coated catheter caused 90% less inflammation in mice and showed more remarkable antibacterial performance. Consequently, this ε-Poly-?-lysine (PL) based coating have a great potential to serve as a safe and multifunctional antibacterial strategy for the medical indwelling devices.