The traditional membrane with single structure cannot satisfy complex clinical applications. Inspired by lotus leaf, a novel structure Janus membrane has achieved more attention recently. Janus membrane is a membrane structure which has two faces with opposite properties. This special structure endows it with asymmetric surface wettability, which can provide an intrinsic driving force to transport along a specified direction, thus achieve unidirectional liquid transport and selective liquid separation. Janus membrane has a promising future, and has been widely used in chemical fields such as self-cleaning, oil/water separation, mist collection, and desalination, while less studied in biomedical field. In this review, the biomedical applications especially in different stages of wound healing process, current challenges in fabrication process and future perspectives of Janus membranes in practical applications under different Janus models, such as hemostasis, bone regeneration, blood cell isolation and gastric mycosal defect will be discussed. It is expected that this unique structure can provide a good therapy prospect in biomedical fields.