Abstract: Three isoenzymes, Cu, Zn-superoxide dismutases I, Ⅱ and III, were isolated from ripe fresh tobacco leaves (K326, Nicotinana) by extraction with 50 mmol/L, pH7.6 phosphate buffer solution (PBS), successive precipitation with 55%-60% (w/w) ammonium sulfate, frozen ethanol-chloroform and acetone, and isolation by DEAE-52 column (balance eluted for 6h with 20mmol/L, pH 7.6 PBS, flow rate 0.25mL/min, then linear gradient-eluted with 20-100mmol/L PBS, flow rate 0.25mL/min) and then by DEAE-52 ion-exchange column (linear gradient-eluted with 30-100 mmol/L PBS, flow rate 0.25mL/min). Pure Cu, Zn-superoxide dismutases I and III were prepared further by G-75 gel column (eluted with 10mmol/L, pH7.6 PBS, flow rate 0.3 mL/min) and freeze-dried at -20℃. It was found that Cu, ZnSODⅠconsisted of 302 amino acid residues, its relative molecular mass was 36682 Da, maximum absorption wavelength was 262 nm, optimal pH value was around 6.0, and optimal temperature was 40℃; Cu, ZnSOD III consisted of 187 amino acid residues, its relative molecular mass was 22976 Da, maximum absorption wavelength was 269 nm, the maximum fluorescence excitation and emission wavelengths were 280 nm and 338 nm, respectively, optimal pH value was 5.0-6.0, and optimal temperature was 30℃.