This study describes a method for isolating two luteolin glycosides from artichokes（Cynara scolymus L.）by preparative liquid chromatography. The partion A27~29 (4 g) of 50% ethanol eluent was achieved by two steps, 70% ethanol extract of artichoke adsorbed by AB-8 macroporous resin and eluted with 20% ethanol, 50% ethanol and 80% ethanol successively. 0.32 g of target part C16~19 with target components H-01 (10.4%) and H-02 (5.1%) was achieved by two steps, A27~29 adsorbed by RP-C18 stationary phase column (270 mm×80 mm, 40~60 μm) and eluted with 30%, 48% and 100% methanol successively. Taking the resolution and shape of target peaks as optimization conditions, the effects of five different mobile phase combinations and two kinds of flow rates on the separation of target peaks were studied. Based on the optimal conditions, H-01 and H-02 with purity of 97.8% and 96.5%, respectively, were obtained. H-01 and H-02 were identified as luteolin-7-rutinoside and luteolin-7-O-β-D-glucoside, respectively, by mass spectrometry, ¹H-NMR and ¹³C-NMR analysis. This method can effectively reduce the impurity level under the condition of high recovery, and provide a new method for the development of the separation and purification process of luteolin compounds.