To improve the oxidative stress tolerance of maize (Zea mays L.), doubled‐haploid (DH) plants were regenerated from in vitro selected microspores exposed to paraquat. The efficiency of in vitro selection was tested by physiological and biochemical investigations, in which the tolerance to paraquat‐mediated oxidative damage and antioxidant capacity of the second generation of fertile microspore‐selected lines (DH2) were determined. In the leaves of paraquat‐selected DH2 lines, reduced accumulation of reactive oxygen species, lower rates of chlorophyll bleaching, protein breakdown and membrane damage, but higher photosynthetic activity were detected on exposure to paraquat stress than either in the leaves of nonselected DH2 lines or in the original hybrid used as controls. The tolerant DH2 plants showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, glutathione S‐transferase, and catalase as compared to both controls. These results suggest that elevated antioxidant capacity may contribute to the paraquat tolerance of the paraquat‐selected DH2 lines and that in vitro microspore selection represents a potential way to improve oxidative stress tolerance in maize.