This paper presents the successful application of ultrasound-assisted packed-bed (UAE-PB) method for the extraction of hypericin from the Hypericum perfuratum L. The Soxhlet system was utilized for the determination of suitable solvent from ethanol, methanol or from the mixture of different proportions of ethanol-methanol. The mixture of 50:50 v/v ethanol-methanol was obtained to be the most suitable solvent since it led to the highest extraction amount of hypericin. The extraction amount of hypericin increased by 13.6% and 21.4% when the solvent changed from pure methanol to the mixture of 50:50 v/v ethanol-methanol for the extraction time of 3 and 8 h, respectively. Subsequently, the extraction was conducted through the UAE-PB, and the effects of temperature, time, and the ratio of solvent to the dried plant were studied. The response surface method (RSM) was used to investigate the effect of parameters on the extraction in the UAE-PB system. At the temperature of 60 °C, extraction time of 105 min, and the solvent to plant ratio of 15.3, the maximum extraction yield of hypericin was achieved. In the optimal conditions, the amount of extraction was 0.112 mg hypericin/g dried plant, which was in accordance with the optimized predicted value (0.111 mg hypericin/g dried plant) from Design-Expert software.