The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of 15N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ~600 mg/L when the yeast cells were cultured in a fermenter. The purified 15N-labeled r-RGD-hirudin retained full biological activity and was uniformly labeled. Heteronuclear NMR of the 15N-labeled r-RGD-hirudin was performed for the first time, and all signals in the heteronuclear single quantum coherence (HSQC) spectrum were successfully assigned.