Introducing unique functional group into proteins is an attractive approach for site-specific protein modification applications. In this study, we systemically investigated four different strategies to introduce azide functionality into recombinant thrombomodulin (TM), site-specifically by recombinant, chemical, and enzymatic methods at either N-terminus or C-terminus for its bio-orthogonal modification application. The first method is to express near C-terminus azido-TM by unnatural amino acid expression. The second method is to incorporate azide molecule into the C-terminus of recombinant TM via sortase-mediated ligation (SML). The third method is to add azide molecule to the N-terminal amine of recombinant TM via amidation chemistry, and the fourth method is to introduce azide to recombinant TMvia a tyrosine selective three-component Mannich reaction. The azido-TMderivatives obtained in all four methods were successfully applied for site-specific modification and immobilization of recombinant TM through copper-free click chemistry with upholding their protein C activation activities. In comparison, incorporating azide molecule into the C-terminus of recombinant TM via SML affords highest overall yield since the key protein expression step uses all natural amino acids. Also, singly site modification and immobilization facilitate the highest TM activity. Overall, the reported site-specific azide incorporation strategies can be widely used in chemical biology and biomedical research and applications.