A chemoselectively surface functionalizable tethered bilayer lipid membrane (tBLM) was developed via liposome immobilization, rupture and fusion processes. Briefly, introduction of triphenylphosphine (TP)-PEG-lipid into a liposome allows it to immobilize onto an azide surface through amide bond formation via Staudinger ligation. Subsequent rupture of the immobilized liposome followed by a second liposome fusion leads to the tBLM formation, which contains TP for further chemically selective modifications on its surface. The membrane fluidity and continuity of the tBLM were confirmed by confocal fluorescence microscopy. The tBLM was covalently functionalized with biomolecules such as azide-containing glycan and biotin in chemically selective fashion and under biocompatible condition, and thus provides a straightforward approach for multifunctional membrane mimetic system fabrication. In addition, the tBLM with incorporated transmembrane protein was demonstrated with an endothelial membrane protein thrombomodulin and its protein C activity was confirmed. The tBLM is very versatile as it can be adapted easily to different types of supporter for a variety of biological and biomedical research areas and applications.