Tumor necrosis factor alpha(TNF-α) is a multi-functional cytokine that plays a significant role in many autoimmune diseases. The key biological functional domain of mouse TNF-α was determined by identifying the binding site of mouse TNF-α neutralizing antibody 9C6. Using yeast surface display technology, it was determined that 9C6 can recognize the linear amino acid fragment 29～40 of mature mouse TNF-α protein. Through mutagenesis experiments of this TNF-α region, the critical amino acids necessary for 9C6 binding were identified. Finally, wild-type mouse TNF-α and mutant variants that loses binding ability to 9C6 were expressed in Escherichia coli, purified, and used in a L929 cell killing assay. The assay results proved that the key amino acids for 9C6 binding were consistent with mouse TNF-α functional domain.