This study aimed to identify the significant factors that give large effects on the efficiency of Cu(II) extraction from aqueous solutions by soybean oil-based organic solvents using fractional factorial design. Six factors (mixing time (t), di-2-ethylhexylphosphoric acid concentration ([D2EHPA]), organic to aqueous phase ratio (O:A), sodium sulfate concentration ([Na2SO4]), equilibrium pH (pHeq) and tributylphosphate concentration ([TBP])) affecting the percentage extraction (%E) of Cu(II) were investigated. A 26-1 fractional factorial design was applied and the results were analyzed statistically. The results show that only [D2EHPA], pHeq and their second-order interaction ([D2EHPA] × pHeq) influenced the %E significantly. Regression models for %E were developed and the adequacy of the reduced model was examined. The results of this study indicate that fractional factorial design is a useful tool for screening a large number of variables and reducing the number of experiments.