Biofunctionalization of poly(ethylene terephthalate) (PET) is crucial to its medical and biomedical applications such as surgical drapes, vascular grafts and ligament prostheses. To furnish PET with an alkynyl handle, N-(2-methylbut-3-yn-2-yl)acrylamide (MBAA) underwent photo-initiated copolymerization with N,N0-methylenebisacrylamide (MBA) in methanol-swollen PET surface to form a 3- dimensional interpenetrating network (IPN). The alkynyl handle terminated surface was denoted as PMBAA-PET. A region-selective modification could be achieved using an engraved mask during the photo-initiated copolymerization. Several functional azides including dansyl-azide 1, azido-5,5- dimethyl-hydantoin analog 2, per-azido-b-cyclodextrin (per-azido-b-CD) and azido-Bovine Serum Albumin (BSA-N3), were successfully bonded onto PMBAA-PET surface via Huisgen 1,3-dipolar cycloaddition. Kinetic study of the heterogeneous “click” reaction between PMBAA-PET and 1 was investigated using X-ray photoelectron spectroscopy (XPS) and elemental analysis. PMBAA-PET was rendered with effective biocidal activity against a healthcare-associated methicilin-resistant Staphylococcus aureus (HAMRSA) and a multi-drug-resistant Escherichia coli (MDR-E. coli) after 2 was conferred. Meanwhile, accessible CD cavity was determined and the amount of covalently immobilized BSA protein was also quantified after the respective “click” linkages of per-azido-b-CD and BSA-N3 on PMBAA-PET surface were established.