Transglutaminase (TGase or TG)， one of the natural food cross-linking agents with strong cross-linking function， form network structures among proteins via ε-(γ-glutamyl)-lysine (G-L) bonds. The gene tgM of TGase was obtained from the genomic DNA of Cordyceps militaris through PCR amplification. It was inserted into an E.coli-P. pastoris shuttle vector pPIC9K to construct recombinant plasmid pPIC9K-TG. The recombinant TGase was successfully expressed in P.pastoris and secreted into the culture medium. In optimized fermentation conditions， the activity of TGase in fermentation broth was up to 100 U/L. The results could provide guidance for the heterologous expression and potential industrial application of TGase.