An ultrasonic-assisted extraction (UAE) procedure of epimedin A, epimedin B, epimedin C and icariin from Herba Epimedii was developed. The effects of ethanol concentration, ratio of liquid to solid, UAE time, extraction temperature and number of extraction cycles on the extraction yields of the four flavonoids from Herba Epimedii were investigated. The optimal UAE condition was found using orthogonal test: 50% (v/v) ethanol solution, liquid:solid ratio of 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 degrees C and three extraction cycles. The UAE method showed a high reproducibility. Epimedin A, B, C and icariin in the crude extract exhibited photodegradation under ultraviolet irradiation. This UAE method was shown to be highly efficient compared with the conventional Soxhlet extraction and boiling extraction. The effect of ultrasound on cell destruction was examined by scanning electron microscopy. The contents of epimedin A, B, C and icariin in the leaves of 20 Epimedium species were determined using high-performance liquid chromatographic method following UAE method.