[Objective] To observe the effects of oxidative stress on the senescence of cardiac fibroblasts (CFb) and the interference effect of 3’-daidzein sulfonate sodium (DSS), to provide an elementary discussion on the mechanism by which 3’-daidzein sulfonate sodium protect myocardium from fibrosis in cardiac senescence. [Method] Cardiac fibroblasts of SD suckling rats were isolated by an initial adhesion step, the cell proliferation ability was determined by MTT assay, NO content of the cell culture supernatant was tested by nitrate reductase method, α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts was detected by immunocytochemical staining, and cell senescence was detected by acridine orange staining. [Result] Under the H2O2 treatment, the proliferation rate of cardiac fibroblasts decreased and immunocytochemical staining of α-SMA was obviously strong positive, the difference is statistically significant compared with control group (P<0.01); NO content was significantly reduced compared with control group (P<0.01); 3’-daidzein sulfonate sodium at three concentration all delayed the above effects induce by H2O2, the difference is statistically significant compared with H2O2 group (P<0.05; P<0.01). [Conclusion] 3’-daidzein sulfonate sodium inhibits the oxidative stress which stimulating the senescence of cardiac fibroblasts, and let cardiac fibroblasts restore the normal proliferation capability. The mechanism underlying may be inhibition of the phenotypic transformation of cardiac fibroblasts by regulating NO and receptor system, which may also be one of the mechanisms contributing to the action of 3’-daidzein sulfonate sodium against myocardial hypertrophy.