Objective The experiment aims at probing the best condition of the isolation and purification of rat islets. Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation. Then the purified islets were subjected to histological staining, electron microscopy and radioimmunoassay for identification of specificity and viability. Results The histological staining revealed that the viability and the purity of the purified islets were above 95% and 85% respectively. Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules. Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly, which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion. There are many factors that influence the quantity and quality of the acquired islets, such as the completed expansion of pancreas, the concentration and viability of collagenase and the digested time, etc.