Abstract: The fatty acids in Eucalyptus urophylla×E. grandis (DH32-29 clone) leaves were extracted by ultrasonic extraction method, and its compositions were analyzed by GC-MS. The D-Optimal response surface methodology were used to optimize eight factors of GC-MS conditions. The content, kinds, Simpson index and TIC of fatty acids were used as responsed value to evaluate the effectiveness of treatment factors. The results of the optimal conditions were as follows:initial temperature 80℃, initial first time (1. 1min) , temperature rate 1∶ 17℃/min) , hold temperature 240℃, hold second time 0. 7min, temperature rate 2∶ 1℃/min, final temperature 253℃, final hold time 4min. Compared with extraction of WC3 leaves from 16 different extraction treatments, the results were also showed that the optimum GC-MS conditions were better than the normal conditions obviously in the content, kinds, Simpson index and TIC of fatty acids.