Using Vitis vinifera as raw material, the effect of main components in polymerase chain reaction (PCR) system on the simple sequence repeats (SSR) amplification of vitis vinifera was studied to optimize dosage of each factor. Total DNA of Grape was extracted by improved hexadecyl trimethyl ammonium bromide (CTAB) extraction method. The influences of the concentrations of main components in SSR-PCR system such as Mg2+, dNTPs, Taq DNA polymerase, primers and the template the vitis vinifera DNA were analyzed through the single factor experiments. Based on orthogonal optimization design, the optimal 25μL reaction system for SSR-PCR of vitis vinifera DNA was established as Mg2+ of 2.5 mmol/L, dNTPs of 0.15 mmol/L, Taq DNA polymerase of 1.5 U, primers of 0.5 μmol/L, and DNA template of 40 ng. Based on visual analysis and variance analysis of optimization experiments, the influences of main components in this PCR system were Mg2+>Taq DNA polymerase>dNTPs>primers>template DNA. 4 kinds of vitis vinifera DNA were amplified using this SSR-PCR amplification system and products were detected using non denaturing polyacrylamide gel electrophoresis. There are clear and stable amplification bands in gels, which indicated that this system could be used to study the SSR mark of vitis vinifera.