The orthogonal design was used to optimize ISSR amplification system of tea plant in five factors (Taq polymerase, Mg2 , DNA templet, dNTP, primer,) at four levels respectively. The data were analyzed by software DPS, which showed that each factor has great effect on the result of PCR; the best level of each factor were selected, a suitable ISSR reaction system was established, namely 20μl reaction system containing 1.0UTaq DNA polymerase, 2.0 mmol/LMg2 , 40ng DNA templet, 0.20 mmol/L dNTP, 0.25μmol/L primer. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR, and found that the most suitable temperature of primer ISSR1, UBC881 was 52.4℃,59.0℃. This optimized ISSR reaction system would provide the basis for the distinction of variety resources, the analysis on genetic diversity, the construction of genetic linkage map etc. by using ISSR markers.