Bulk nanocrystalline alloys usually possess enhanced properties than their coarse-grained counterparts. Here, first-principles calculations and aberration-corrected transmission electron microscope (TEM) were employed to investigate the atomic-scale deformation mechanism of Cu-based alloys. The effect of alloying element concentration and temperature-induced solute distribution on the unstable stacking fault energy (γusf), stable stacking fault energy (γisf) and unstable twin fault energy (γutf) were calculated using a Fermi–Dirac distribution of solutes for 42 binary Cu-X alloys. At medium temperature (>200 K) or low solute concentrations (<15 at.%), the stacking fault energies calculated from the Fermi–Dirac model accord well with the available experimental and theoretical results. The deformation mechanism was then evaluated by α = γisf/γusf and β = γutf/γusf, smaller α (β) favors an easier formability of extended dislocations (twins). Most subgroup VI-VIII metals in the periodic table can slightly increase the γusf, γisf and γutf of Cu, and have almost no influence on α and β. While main group and subgroup II-V elements can decrease γusf, γisf and γutf as well as the values of α and β. For alloying elements of Pd, Ag, Pt and Au, the values of α and β increase, suggesting a tendency of deformation mechanism from extended dislocations to full dislocations. Furthermore, high-resolution TEM (HRTEM) images of four representative nanocrystalline alloys (pure Cu, Cu-Fe, Cu-Ag and Cu-Zn) corroborates the prodiction with α and β as well as the empirical twinnability. The α and β remain almost the same as that of pure Cu when alloyed with Fe while they decrease with Zn, and the extended dislocations and twins were commonly observed for Cu, Cu-Fe and Cu-Zn. The α and β increased with Ag addition although the γisf decreased, and the extended dislocations were barely observed for Cu-Ag sample. The theoretical and microstructural correlation provides insights into the deformation mechanism of Cu-based alloys.