Emilia prenanthoidea DC. (EP) is a medicinal plant that belongs to the tribe Emilia Cass in the family Asteraceae. Although it has a long history of medicinal use, there are few research reports on this herb. In this study, for the first time, ultrasound-assisted extraction (UAE) conditions were optimized by response surface methodology (RSM) to extract total flavonoids from EP. An optimized method was used to determine the total flavonoid content (TFC) and antioxidant activity of EP extracts from 12 regions in Guizhou, China. Combining environmental factors in 12 regions, the effects of different growth environments on TFC and antioxidant activity of EP extracts were analyzed. In this work, it is proven that the optimal conditions for extraction are a solvent concentration of 45%, an ultrasonication time of 25 min, and a liquid-solid ratio of 46.25 mL/g. Under this condition, the highest TFC (41.182 mg/g) was found in the EP extract from Daping, while the lowest TFC (28.865 mg/g) was found in the EP extract from Liping. Evaluation of antioxidant activity showed that the highest antioxidant capacity was found in EP extracts from Daping, followed by Tuan Shan, Yaobai and Green Lake. Ecological factors affected both total flavonoids and antioxidant activity in EP. Annual sunshine hours in different regions had a significant effect on TFC in EP (r = −0.841, P < 0.01). In conclusion, this study established an effective method for the extraction of total flavonoids from EP, an effective natural antioxidant. The effects of the growth environment on the TFC and antioxidant capacity were also analyzed. It provides an experimental basis for the extensive utilization of EP.