Glucagon-like peptide-1 (GLP-1), is currently used to treat type 2 diabetes mellitus and hirudin (HV), plays an important role in controlling thrombosis and cardiovascular diseases. This investigation aimed to develop a fusion peptide 5rolGLP-HV which combined functions of rolGLP-1 and rHV to treat diabetes and thrombosis. In this study, we constructed a fusion gene including five copies of rolGLP-1 and one copy of rHV (5rolGLP-HV). The optimum expression conditions of 5rolGLP-HV in a soluble form were 0.8 mM IPTG induction when OD600 reached 0.6-0.8 and further growing at 25 A degrees C for 9 h. Isolated rolGLP-1 and rHV were acquired by trypsin digestion in vitro, and the concentration of them was determined by HPLC in vivo. Oral administration of 5rolGLP-HV significantly decreased the levels of blood glucose, GHbA1C, TC, and TG in diabetic mice at the time of 3 weeks compared to the saline-treated group (p < 0.05), while the insulin level was reversed significantly (p < 0.05). 5rolGLP-HV treatment significantly shortened the length of thrombus in thrombosis mice compared to the saline-treated group (p < 0.01). These results indicated that 5rolGLP-HV had dual-function in treating diabetes and preventing thrombosis.