Abstract: p-Azido-L-phenylalanine(pAzPhe) hydrochlorade with high purity was prepared by five steps using p-nitro-L-phenylalanine as the starting material.Recombinant expression vector was constructed to express human endostatin mutant rhEs31m.Then pAzPhe was site-specifically (site 31) incorporated into recombinant human endostatin (rhEs) by applying the optimized orthogonal aminoacyl tRNA synthetase/tRNA pair.The products after purification were identified by SDS-PAGE and Western blotting.The purity of the mutant is beyond 90%,and can be specifically recognized by antibody of human endostatin.The HUVEC(human umbilical vein endothelial cell) proliferation in vitro by rhEs31m was analyzed by MTT,and results revealed that rhEs31m retained most of the bioactivity of wild type rhEs.The study indicates that site-specific incorporation of single p AzPhe does not destroy the spatial structure and bioactivity of rhEs.