Copper oxide nano materials (NMs) effects compared to macro materials (MMs) was investigated on physiological and gene expression of rue plant. Treatments of 0.1, 0.5 and 2.5 µM of copper with three replications were applied to plants under hydroponic conditions. Copper absorption decreased according to MMs-CuO ˂ NMs-CuO ˂˂ CuSO4 pattern. The positive effects of MMs-CuO on plant copper content were higher than those of nano- and CuSO4. MMs-CuO effect was more significant on plant biomass increase compared to the control. Rue plant needed lower amounts of copper for better plant growth. The treatments increased protein and carotenoids content in leaves compared with control. Changes in total chlorophyll content under three copper forms was very low and was only increased in leaves at 0.1 µM CuSO4. The NMs-CuO and MMs-CuO induced similar effects, and reduced leaves’ Cu, MDA and ROS content, and SOD activities. CAT enzyme activity had a similar pattern in three copper forms. CAT enzyme activity was only induced under the lowest level of three forms, while at other levels of Cu it was reduced. NMs-CuO had a more negative effect on IRT1 relative gene expression in root compared with other iron forms. The IRT1 relative gene expression in shoots was positively affected under 2.5 µM CuSO4, 0.5 µM MMs-CuO, and 0.1 and 2.5 µM NMs CuO treatments. This study could better elucidate micro and nano CuO effects on physiology and gene expression mechanisms in rue plants.