Objective To construct yeast eukaryotic expression vector carrying human endostatin (ES) cDNA. Methods The functional fragment of endostatin gene in human hepatic tissue was amplified by using RT-PCR technology, and cloned into yeast pPIC9 expression vector. The positive clone was sequenced by using automatized sequencer. Results The endostatin cDNA was successfully cloned. The positive ES clone gene in pPIC9 expression vector was sieved, and its coding sequence was identified to be as same as the previously reported sequence. Conclusion The successful construction of ES gene in pPIC9 expression vector using molecular biological method maybe helpful for the high expression of ES protein, which may lay the foundation for the treatment of malignant tumor through anti-angiogenesis appoach.