To explore the effects of metal nanoparticles on plant growth, agar culture simulating soil was used to characterize the root elongation and relative physiological and biochemical behavior of wheat (Triticum aestivum L.) under stress of copper oxide nanoparticles (CuO NPs). The results showed that the inhibition of root elongation in agar was exponentially correlated with the concentration of CuO NPs, which was increased slightly at a lower concentration (10 mg/L) of CuO NPs but decreased greatly at a higher concentration (100 mg/L). It should be noted that the variation of superoxide dismutase (SOD) activity shared the same trend as the inhibition of root elongation under CuO NPs exposure. Furthermore, with increasing of CuO NPs from 1 to 100 mg/L, the content of malonaldehyde (MDA) continuously increased while the protein content sharply decreased in roots. These results demonstrated that the inhibition of root elongation could be mainly attributed to the peroxide injury of plant cell membrane under CuO NPs exposure. Interestingly, wheat root may exhibit the adaptive response through enhancing the root vitality to alleviate the phytotoxicity induced by CuO NPs.