The main objective of this work was to purify the exopolysaccharides (EPS) of Flammulina velutipes SF-06 and investigate the relationship between the different purified fractions and bioactive activity. Two fractions (EPS-1 and EPS-2) were separated and purified by DEAE-52 cellulose and Sephadex G-100 cellulose column chromatography. Monosaccharides composition analysis by gas chromatography indicated that EPS, EPS-1 and EPS-2 were heteropolysaccharides in which rhamnose was a major component. Fourier transform infrared (FT-IR) analysis detected furanose-ring in EPS-1 and EPS-2. All fractions possessed considerable antioxidant activity, while EPS-2 has stronger antioxidant activity than EPS and EPS-1 in vitro. The EPS also exhibited potent anti-aging activation in mice, such as increased catalase and total antioxidant capacity, and decreasing the malondialdehyde (MDA) content. Both the antioxidant in vitro and anti-aging in vivo potentials of EPS could be further utilized in the food industry.