The binding characteristics and superimposed antioxidant properties of caffeine combined with copper/zinc superoxide dismutase (SOD) were studied. The superimposed antioxidant activity of caffeine with SOD was investigated by detecting the concentration of malondialdehyde (MDA) present in cells, which was induced by hyperthermia and heavy metal exposure. The interactions between the SOD enzyme and caffeine were researched by ultraviolet spectrum, fluorescence spectrum, and molecular computation. The relative amounts of MDA contents of caffeine (0.1 mmol/L), SOD (0.1 mg/L), and caffeine (0.1 mmol/L) and SOD (0.1 mg/L) to water in cells were 0.70, 0.72, and 0.54, respectively, indicating that the antioxidant properties of caffeine combined with SOD may be superimposed. The fluorescence spectroscopy and molecular computation results show that the mixture of caffeine and SOD can result in the formation of a 1:1 complex through hydrogen bond and van der Waals forces spontaneously. The binding constant ( K a ) of caffeine with SOD at five different temperatures are 4.41 × 10 4 , 3.30 × 10 4 , 2.29 × 10 4 , 1.71 × 10 4 , and 1.17 × 10 4 L/mol. The changes of Gibbs-free energy (Δ G ) are -26.50, -26.21, -25.71, -25.12, and -24.29 KJ/mol and the Δ G of molecular docking calculation is -26.95 KJ/mol. The experimental results are in accordance with the results of theoretical calculations. The combination of caffeine with SOD can change the conformation and microenvironment of SOD but does not change the activity of SOD. In addition, the combination can superimpose the antioxidant activity of caffeine and SOD., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)