This study was aimed to prepare a live attenuated vaccine by a new method from local methicillin-resistant Staphylococcus aureus (MRSA) isolate as a trial to control bovine mastitis. The isolated MRSA was confirmed by PCR, then live attenuated vaccine was prepared by a new method using 3% H2O2 followed by serial passage thirty times. Wistar albino rats were used as an experimental animal model to estimate the immune response against prepared vaccine. ELISA assay was used to detect CD4+ and CD8+ T-cells concentrations while RT-qPCR was used to detect IL-2 and IFN-γ gene expression to estimate immune response results from vaccination by the prepared vaccine. Results showed a significant increase both in CD4+ and CD8+ T-cells concentrations using ELISA. Also, RT-qPCR showed up-regulation of each of IL-2 and IFN-γ gene expression by increasing in change folds reached to 2612.11 and 704.28 times respectively for both of them. CD4+ T-cells can enhance the humoral immune response by differentiation to Th2 cells and cell-mediated immunity (CMI) by differentiation to Th1 cells, in addition, to enhancing effect of IFN-γ through a positive feedback mechanism that amplifies the Th1 effect. CD8+ T-cells differentiated to Cytotoxic T Lymphocytes (CTLs) that are responsible for cell-mediated immunity by the killing of infected cells by intracellular pathogens, in addition to IL-2 that enhance the effect of CTLs also. It concluded that prepared live attenuated vaccine stimulated both humoral and potent cell-mediated immunity promising it as a good new method for live attenuated vaccine production.