Graphene oxide (GO) is a widely used nanomaterial, whose unique carbon-based structure bears abundant oxygen-containing functional groups. An increasing number of investigations have been performed on the effects of GO on plants due to its extensive use in various fields. In this work, under the optimized GO concentration (6 mg/L), we found that GO promoted the growth rate of the root of poplar. TEM images depicted that the plasmolysis and an increased number of starch grains were the two major physiological changes in the root cell samples. GO adhered to the cell membrane on the inner side of the cell wall and enhanced the toughness of the adventitious root. And the activity of oxidoreductases were measured and SOD, POD were found to increase significantly compared with the control group, but the increase of CAT was not obvious. Related gene expression was enhanced at low concentration of GO, but inhibited at high concentration. Our results indicate the possible usage of GO as enhancers in the growth of poplar tissue culture seedlings.