Objective： To optimize the extraction process of Mistletoe by using the Box-Behnken design-response surface method, and the comprehensive content of the two active ingredients as the evaluation index. Methods: The ethanol volume fraction, extraction time and solid-liquid ratio of Mistletoe extraction process were optimized by using the Box- Behnken design-response surface method on the basis of single factor. Ultra performance liquid chromatography (UPLC) was used to determine the content of two active components in Mistletoe from 14 origins. Results: The optimal extraction process of Mistletoe was 71% ethanolaqueous solution, liquid-solid ratio 25:1, and extraction time 67 min. The optimal extraction process showed that the average comprehensive content was 571.7 μg·g-1 when the weight ratio of Syringin to homoeriodictyol-7-O-β-D-glucoside was 7:3. The contents of Syringin and homoeriodictyol-7-O-β-D-glucoside ranged from 128.7~662.8 μg·g-1 and 77.24~801.8 μg·g-1. The contents of Syringin and homoeriodictyol-7-O-β-D-glucoside in Mistletoe from different origins were different, among which the content of homoeriodictyol-7-O-β-D-glucoside in Mistletoe from Sichuan was relatively high, which was 0.84 mg·g-1, and the content of Syringin from Mistletoe from Anhui was relatively high at 0.47 mg·g-1. Conclusion: The method established in this study has high sensitivity, good separation effect, stable and reliable results, which can provide a scientific basis for the extraction of active ingredients, quality evaluation and resource development and utilization of Mistletoe.