Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and is implicated in the evasion of the host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can illuminate the biological functions of ISG15 and inform related disease interventions, but such studies necessitate the expedient preparation and development of various ISG15 protein tools. Here, we report a finding that the leader protease (Lb pro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, enabling efficient preparation of various ISG15 protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, the Lb pro ligation strategy can also be used for loading recombinant ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and for chemo-enzymatic preparation of Ub and NEDD8 protein tools.