Abstract: Various methods including multiple column layers ,TLC, HLPC,UV and MS were adopted in the isolation，purification and identification of the growth promoting substance in the fermented liquid of Trichoderma harzianum T2-16. The test result suggested the growth promoting substance was a kind of peptide. The effect mechanism of growth promoting substance was studied. The effect on nodule microstructure and nitrogen-fixing of cowpea were studied through it was applied by seed soaking in greenhouse pot tests. The results as follows: The ratio of center tissue (tissue infected) and the cells in the nodules of cowpea treated by growth promoting substance had much bigger and less vacuoles than those in treated by water. The number of bacteroid in per cell and the content of leghaemoglobin of cowpea nodule treated by growth promoting substance was also remarkably increased. Meanwhile, the common peribacteroid membranes were appeared in a high frequency than those treated by water. The results showed that cowpea treated by growth promoting substance had higher production of leghaemoglobin and different microstructure and bacterioid ultrastructure of cowpea nodule which led to a highter nitrogenase activity of peanut nodule .