Anhydrosafflor yellow B (AHSYB) is a major active water-soluble pigment in Safflower, but it has not received enough attention yet. In this study, high-speed counter-current chromatography (HSCCC) was used to prepare AHSYB from safflower. The parameters of the separation process were optimized by response surface methodology for the first time. The entropy weight method (EWM) was applied to calculate the information entropy and the weight of five indexes, and then figure out a comprehensive index of the HSCCC separation effect. Under the optimized separation conditions, a HSCCC apparatus speed of 850 rpm, a flow rate of 2 mL min⁻¹ for the mobile phase and a separation temperature of 40 °C for AHSYB were achieved with a purity of 98%. Furthermore, AHSYB was found to have cardio-protective effects by inhibiting apoptosis via the mitochondrial-mediated pathway in oxygen-glucose deprivation/reoxygenation-induced H9c2 cells. This research provides good method guides for the rapid and efficient separation of active compounds from food-grade Chinese herb medicines.