Abstract: Objective:To optimize the extraction technology of total flavonoids from Anoectochilus roxburghii,the uniform design method was used and the total flavonoids contents of A. roxburghii of different months and various Anoectochilus spp were compared. Method:Uniform design method was applied to investigate the factors such as ethanol concentration,liquid material ratio,extraction time and extraction rate on total flavonoids extraction of A. roxburghii. The optimized extraction method from uniform design method was then validated by determined the flavonoid contents of different months A. roxburghii and other Anoectochilus spp. Results:Optimized extraction technology was found to be 40 volumes of 88% ethanol,80 min ultrasonic extraction,and 3 times of repetition. The total flavonoids extraction rate in verification test was 1.68%(RSD=0.27%,n=3). The total flavonoid contents of A. roxburghii was correlated to its strain and month of ages. The average contents of total flavonoids in A. roxburghii tissue cultured was 11.091 mg/g,The average contents of total flavonoids in 4 and 5 cultivated month age of cultivated A. roxburghii were 14.359 and 18.730 mg/g,respectively. The amount of total flavonoids obtained from the 4 cultivated month age of A. roxburghii arranged in decreasing order as Da Yuanye > Jian Ye > Hong Xia > Local Jianye > Local Yuanye > Local Wild. The finding also suggested that the secondary metabolites contents especially flavonoid compound in cultivated A. roxburghii was believed to be increased gradually,and reached the highest value at the age of 5 month. Conclusion:The optimized ultrasonic extraction process was reasonable,stable,and feasible,would provide a reference for the determination and quality evaluation of total flavonoids in A. roxburghii. There had differences between tissue culture and cultivated A. roxburghii in the contents of total flavonoids. The soil cultivation was beneficial to the accumulation of the active constituents of A. acuminata. Therefore,it was necessary to cultivate the golden lotus seedlings through soil cultivation.