A highly sensitive and selective catalytic kinetic spectrophotometric method for the determination of Cu (II) is proposed. It is based on the catalytic effect of Cu (II) on the oxidation of glutathione (GSH) by potassium hexacyanoferrate (III) in acidic medium at 25.0℃. The reaction is monitored spectrophotometrically by measuring the decrease in absorbance of oxidant at 420 nm using the fix-time method. Under the optimum conditions, the proposed method allows the determination of Cu (II) in a range of 0 - 35.0 ng mL −1 with good precision and accuracy and the limit of detection is down to 0.04 ng mL −1. The relative standard deviation (RSD) is 1.02%. The reaction orders with respect to each reagent are found to be 1, 1/2, and 1/2 for potassium hexacyanoferrate (III), glutathione and Cu (II) respectively. On the basis of these values, the rate equation is obtained and the possible mechanism is established. Moreover, few anions and cations can interfere with the determination of Cu (II). The new proposed method can be successfully used to the determination of Cu (II) in fresh water samples and seawater samples. It is found that the proposed method has fairly good selectivity, high sensitivity, good repeatability, simplicity and rapidity.