Abstract: OBJECTIVE To characterize the molecular weight of Radix Isatidis protein (RIP) and optimize the extraction process of protein from Radix Isatidis. METHODS Response surface methodology (RSM) was applied to optimize the extraction of protein from Radix Isatidis. Extraction time， liquid-to-solid ratio and pH value were set as the investigated factors with respect to the protein yield. In addition， Design Expert software was used for data analysis. The RIP was characterized for composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Scanning electron microscopy (SEM) analysis was performed to observe microstructure of the Radix Isatidis powder before and after ultrasound-assisted extraction (UAE). RESULTS Based on the RSM analysis， optimum conditions were determined as follows: twice ultrasonic extraction in 50 mmol/L Tris-HCl buffer solution， pH at 7.8， liquid-to-solid ratio at 80∶1 and extraction for 65 min each time. Under the optimized conditions， the experimental values were 0.705%， which is in close agreement with values predicted by the model. The characterization of the RIP demonstrated that it contained five major groups of protein bands， namely bands of 19.2 kDa， 21.5 kDa， 24.8 kDa， 34~43 kDa and >170 kDa respectively. CONCLUSION RSM can be applied for the optimization of extraction process of RIP， which is effective， stable and feasible.