We carried out pot experiments to explore the effects of graphene oxide (GO) added in soil with different dosages (1%, 2%, 3%, 4% and 5%) on the growth and physiological characteristics of stress resistance in ryegrass (Lolium perenne L.) roots. The results showed that: (1) the root growth was not affected by low dosages of GO (1% and 2%), but it was significantly inhibited by high dosages of GO (3%-5%). (2) No significant differences were observed in root volume and biomass between 1% GO treatment and the control. With the increase of GO dosage, root volume and biomass of ryegrass decreased. Root dry weight was the lowest under 5% GO treatment, which was decreased by 22.64% compared with the control. (3) 1% GO treatment did not affect the activities of protective enzymes and malondialdehyde (MDA) content. The activities of POD, SOD and CAT enzymes was higher than that of the control with increasing GO dosage, and MDA content also increased significantly. (4) When the dosage of GO exceeded 1%, the root activity of ryegrass significantly decreased. The lowest value was recorded under 5% GO treatment, which was 35.07% lower than that of the control. The findings suggest that low dosages of GO could not affect root growth of ryegrass, while high dosages of GO could induce oxidative stress on roots, resulting in root damage.