In order to discuss the extraction and separation mechanism of copper and nickel by 1-phennyl-1,3-sebacic-diketone (Mextral54-100) from ammonia solution, the binding energy, global activity indices, local activity indices and infrared spectrum (FT-IR) of copper complexes and nickel complexes were calculated by density functional theory DFT/B3LYP/6-31G+(d, p), based on quantum chemistry. The results showed that the extracting power of copper was beyond nickel with Mextral54-100. Global reactivity indexes pointed out that the order of the activity of the copper complexes was a>b>c, the order of the activity of the nickel complexes was d>e>f. The molecule frontal orbital analysis of the complexes indicated that the |Δe| (Δe=eHOMO?eLUMO) of a was |Δe|a=0.158, the |Δe| of d was |Δe|d=0.138, thus, the reaction activity of a was higher than that of d, and the copper complexes were easier than nickel complexes in the stripping process by the theoretical calculation results. Local activity indices showed out that the carbonyl exhibited the highest reactivity and was the active center. The more substitution number of ammonia molecules in the complexes, the stronger electron-donating ability of copper(II) and nickel(II), and the structure of the complex was similar to the stable octahedral structure. The ammonia molecules were replaced one by one by the carbonyl group in extractants, avoiding the co-extraction of ammonia effectively. The experimental results of extraction and stripping of copper and nickel from ammonia solution by Mextral54-100 were in good agreement with the theoretical calculation results by DFT, and which could be verified the accuracy of theoretical calculation results by FT-IR. What was more, DFT was expected a novel method to study the extraction and separation mechanism copper and nickel from ammonia solution in the future.