Streptomyces transglutaminase (TGase) has been widely used in food, pharmaceutical and textile industries. Streptomyces TGase is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by removing its N-terminal pro-peptide. Although the pro-peptide is essential for TGase folding and secretion, few studies have been reported on improving the properties of TGase by pro-peptide engineering. In this study, we developed a new approach to improve the properties of TGase based on pro-peptide engineering. When the α-helix(37G-42S) in pro-peptide was substituted with three glycines and three alanines respectively, the mutants exhibited higher specific activity and the efficiency of pro-peptide cleavage was enhanced. To further improve the properties of TGase, relevant mutations were constructed by introducing linker peptides in the C-terminus of the pro-peptide. Mutants with GS (GGGGS) and PT (PTPPTTPT) linker peptide exhibited 1.28 fold and 1.5 fold higher specific activity than the wild-type enzyme, respectively. This new method could be used to improve the properties of TGase by pro-peptide modification, which is a promising technology for creating unique TGase with various beneficial properties.