Flavonoids are secondary metabolites of plants that often have medical applications. The influences of different sample drying pretreatments on flavonoids and antioxidant activity of ferns have not studies. Dryopteris erythrosora leaves used to analyze flavonoid alterations resulting from drying pretreatments. The total flavonoid content of D. erythrosora leaves exposed to different pretreatments was significantly different. The total flavonoid content of samples initially air-dried in shade and then oven-dried at 75C were the highest (7.6%), while samples initially dried at 75C had the lowest content (2.17%). Antioxidant activities of D. erythrosora leaves with different pretreatments varied. Group B first air-dried in the shade and then oven-dried at 75C and group C first air-dried in the sun and then oven-dried at 75C, both showed relatively stronger antioxidant activity. The best pretreatment for preserving the flavonoids was to first dry the plant material in the shade and then complete the drying process in an oven at 75C. It was tentatively identified 22 flavonoids among the four different pretreatments by HPLC-ESI-TOF-MS.