Graphene nanomaterials have many diverse applications, but are considered to be emerging environmental pollutants. Thus, their potential environmental risks and biosafety are receiving increased attention. Bioaccumulation and toxicity evaluations in plants are essential for biosafety assessment. In this study, 13C-stable isotope labeling of the carbon skeleton of graphene oxide (GO) was applied to investigate the bioaccumulation and toxicity of GO in wheat. Bioaccumulation of GO was accurately quantified according to the 13C/12C ratio. Wheat seedlings were exposed to 13C-labeled GO at 1.0 mg/mL in nutrient solution for 15 d. 13C-GO accumulated predominantly in the root with a content of 112 μg/g at day 15, hindered the development and growth of wheat plants, disrupted root structure and cellular ultrastructure, and promoted oxidative stress. The GO that accumulated in the root showed extremely limited translocation to the stem and leaves. During the experimental period, GO was excreted slowly from the root. GO inhibited the germination of wheat seeds at high concentrations (≥0.4 mg/mL). The mechanism of GO toxicity to wheat may be associated with oxidative stress induced by GO bioaccumulation, reflected by the changes of malondialdehyde concentration, catalase activity, and peroxidase activity. The results demonstrate that 13C labeling is a promising method to investigate environmental impacts and fates of carbon nanomaterials in biological systems.