The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys18) salmon calcitonin (Lys18-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC1,11-sCT), which was FMOC protected at Cys1- and Lys11-amines among three PEGylation sites including Lys18-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC1,11-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC1,11-sCT. The optimized reaction resulted in the high production yield of Lys18-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys18-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys18-PEG-sCT conjugate with a high yield.