Iridoid glycosides were extracted from Gentiana rhodantha Franch. by microwave-assisted aqueous two-phase system. The optimal extraction process was determined by single factor experiment and orthogonal experiment. The total antioxidant activity of iridoid glycosides and their ability to scavenge 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals were evaluated. The results showed that the optimum extraction conditions were obtained as follows: ethanol 7.8 g (mass fraction 26%) and K2HPO4 3.0 g (mass fraction 10%) in the establishment of 30.0 g ethanol/K2HPO4 aqueous two-phase system, particle size of dry Gentiana rhodantha Franch. powder 400 mesh, microwave extraction temperature 70±1℃, microwave extraction time 110 s and dry powder 3.5 g. Under these conditions, the extraction yield of iridoid glycosides was 5.48%, higher than that obtained by traditional extraction process (3.61%). This method can inhibit the activity of endogenous hydrolase in the test material and neutralize phytic acid (the acid substance in Gentiana rhodantha Franch.) to reduce the polymerization of dissolved iridoid glycosides, which effectively solves the problem that the iridoid glycosides in Gentiana rhodantha Franch. must be enriched to be detected. Total iridoid glycosides had stronger antioxidant activity than 2,6-di-tert-butyl-4-methylphenol (BHT) in the range of 0.2~0.6 g/L, and stronger scavenging activity to DPPH radicals than BHT in the range of 0.2~1.0 g/L.