The aim of this paper is to optimize the EST-SSR system on heading Chinese cabbage, and select a certain amount of primer pairs using the optimized system. L16(44) orthogonal design is used in the system optimization, and the concentrations of the main factors are studied to build the best system. The results show that the different levels of each factor have some effects on the results of EST-SSR, with the effect of the primer concentration being the greatest, those of dNTP and Taq DNA polymerase the next, and that of the concentration of DNA template the minimal. The optimized system(20?滋L) is: 2.0?滋L 10×PCR Buffer with Mg2+, 70ng DNA template, 250?滋mol/L dNTP, 1.0?滋mol/L primer, and 0.5U Taq DNA polymerase. One hundred and twenty-six primer pairs are designed according to the EST sequences of heading Chinese cabbage, and EST-SSR PCR is amplified by using the above primer pairs and optimized system, with nineteen primer pairs being selected based on their reproducible and polymorphism, which could be used for further research.