Hydrogen peroxide (H2O2)-induced hemolysis is a commonly used model for antioxidant activity evaluation, and the hemolysis index is often presented as the absorbance of supernatant hemoglobin (Hb), which releases from injured cells. However, in previous studies, as an oxidation-sensitive protein, it has been recognized that Hb easily forms other types or substances, such as metHb, Heinz, and some fluorescent products. This study concerns whether Hb oxidation participated in H2O2-induced hemolysis and confirmed that the destruction of Hb under oxidizing condition had been a novel interfering factor that could reduce the absorbance in Hb quantitative detection. To correct the lower absorbance, the stable fluorescent products found in Hb degradation were selected, and the absorbance correction factor of 6.436 was drawn on the basis of the absorbance and fluorescent intensity. This correction factor obviously altered the results of both dose-dependent hemolysis of H2O2 and antioxidant activity. In addition, the assessment difference was innovatively discussed by altering the sequences of adding antioxidant and oxidant. These different sequences caused variations in hemolysis, indicating that multiple evaluations may be related to the antioxidant pathways which are necessary for more accurate bioactivity data.