To investigate the effects of ozone on winter wheat and flixweed under competition, two species were exposed to ambient, elevated and high [O3] for 30 days, planted singly or in mixculture. Eco-physiological responses were examined at different [O3] and fumigating time. Ozone reduced the contents of chlorophyll, increased the accumulation of H2O2 and malondialdehyde in both wheat and flixweed. The effects of competition on chlorophyll content of wheat emerged at elevated and high [O3], while that of flixweed emerged only at high [O3]. The increase of H2O2 and malondialdehyde of flixweed was less than that of wheat under the same condition. Antioxidant enzyme activities of wheat and flixweed were seriously depressed by perennial and serious treatment using O3. However, short-term and moderate fumigation increased the activities of SOD and POD of wheat, and CAT of flixweed. The expression levels of antioxidant enzymes related genes provided explanation for these results. Furthermore, the increase of CAT expression of flixweed was much higher than that of SOD and POD expression of wheat. Ozone and competition resulted in significant reductions in biomass and grain yield in both winter wheat and flixweed. However, the negative effects on flixweed were less than wheat. Our results demonstrated that winter wheat is more sensitive to O3 and competition than flixweed, providing valuable data for further investigation on responses of winter wheat to ozone pollution, in particular combined with species competition.