In this study, secondary metabolites of Eurotium cristatum were isolated and purified by high-speed counter-current chromatography (HSCCC), and their hypoglycemic activities were studied. The general-useful estimate of solvent systems (GUESS) for counter-current chromatography was employed to select the appropriate solvent systems of n-hexane-ethyl acetate-methanol-water (HEMW, 4:6:5:5, v/v/v/v) for HSCCC practice, and three compounds were separated from the crude ethyl acetate extract of Eurotium cristatum in one single step. 6.1 mg of Compounds 1, 5.6 mg of Compound 2 and 3.8 mg of Compound 3 were obtained from 100 mg of crude extract with a stationary phase retention of 75%. The compounds were then identified as emodin methyl ether, chrysophanol and emodin, respectively. The activity of the target compounds in the secondary metabolites of Eurotium cristatum was verified by testing their inhibition on α-glucosidase activity and molecular docking simulation. The results showed that emodin, chrysophanol and emodin methyl ether had significant inhibitory effects on the α-glucosidase activity. This work confirmed the effectiveness of HSCCC in the separation of compounds in complex extracts, and provided reference for further research and application of Eurotium cristatum.