Raffinose family oligosaccharides (RFOs) were extracted and isolated from Rehmannia glutinosa Libosch. by using microwave-assisted extraction and fast protein liquid chromatography coupled with refractive index detection (FPLC-RID). After extraction with 60% ethanol (v/v) and decolorization with MCI gel CHP20P, total oligosaccharides were purified by using FPLC-RID coupled with the Bio-Gel P2 column eluted with water at the flow rate of 0.3 mL/min. Three pure oligosaccharides (DP3-DP5) were obtained with the yields of 1.72%, 7.52% and 0.51%, respectively, as well as identified as raffinose, stachyose and verbascose from the results of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS) and gas chromatography coupled with mass spectroscopy (GC-MS) analysis. A large-scale of pure RFOs was automatically and efficiently separated from R. glutinosa by using FPLC-RID method, which was beneficial for the qualitative and quantitative analysis of RFOs in R. glutinosa and their products.