Worldwide, impaired wound healing leads to a large burden of morbidity and mortality. Current treatments have several limitations. Recently, nanomaterials such as copper nanoparticles (CuNPs) have attracted considerable research interest. Here, we investigated the potential therapeutic effect of various CuNPs concentrations (1 μM, 10 μM, 100 μM, 1 mM, and 10 mM) and sizes (20 nm, 40 nm, 80 nm) in wound healing. Our results revealed that the 10 μM concentration of 40 nm CuNPs and the 1 μM concentration of 80 nm CuNPs were not toxic to the cultured fibroblast, endothelial, and keratinocyte cells, and also 1 μM concentration of 80 nm CuNPs enhanced endothelial cell migration and proliferation. Extensive assessment of in vivo wound healing demonstrated that the 1 μM concentration of 80 nm CuNPs accelerated wound healing over a shorter time via formation of granulation tissue and higher new blood vessels. Importantly, serum biochemical analysis confirmed that the 40 nm CuNP (10 μM) and 80 nm CuNP (1 μM) did not show any accumulation in the liver during wound healing. Overall, our results have indicated that the 1 μM concentration of 80 nm CuNPs is a promising NP for wound healing applications without adverse side effects.