Abstract: OBJECTIVE The method of simultaneous hydrolysis and extraction in situ was applied to process 80% of the volume fraction of ethanol extract of Acanthopanax senticosus, in order to convert the bound isofraxidin into free isofraxidin. METHODS The optimum conditions were confirmed as follows: the hydrochloric acid concentration of 0.6 mol·L^-1, the ratio of solid to liquid was 1∶100 by adding hydrochloric acid, the same volume of 1,2-dichloroethane for extraction，reflux temperature 85 ℃, hydrolysis time 2.5 h. RESULTS Under these conditions, the isofraxidin content in the root bark, stem bark and root-stem mixture product had increased from 0.042 4, 0.011 2 and 0.005 3 mg·mL^-1 to 1.264 1, 0.441 2 and 0.260 0 mg·mL^-1, which were 28.81-fold, 38.39-fold and 48.06-fold of those in the untreated extracts, respectively; the isofraxidin content in the root xylem and stem xylem were 0.023 3 and 0.009 7 mg·mL^-1, which can't detect the concentration without treatment. CONLUTSION Using the process of simultaneous extraction and hydrolysis for separation of isofraxidin, hydrolysis temperature decreased from 100 ℃ to 85 ℃, hydrolysis time shortened from 14.5 h to 2.5 h and the solvent amount was only the three-quarter of traditional method, comparing with the traditional acid hydrolysis and then extract with organic solvents method.