The AtEIN3 gene of Arabidopsis thaliana was used as probe and its high homology sequences which were screened from TM1 of cotton data base were analyzed in this study. 16 sequences with EIN3 domain were obtained through local Blast alignment of Cottongen to determine the intron and exon, phylogenetic relationship, sequence similarity and conserved motif distribution. The Blast results showed that the cottons EIN3/EIL and the A. thalianas had high similarity at the Nterminal, and 15 of them contained 5 motifs, 1 including 4 motifs. A EIN3/EIL gene was cloned from the roots of ‘zhongmiansuo 12’ which was Fusarium Wilt Resistance cotton cultivar by RTPCR method, named GhEIL3（GenBank accession number KY072936）according to its relationship with the evolution of A. thaliana. Sequence analysis showed that the open reading frame (ORF) of GhEIL3 was 1 092 bp, encoding 363 amino acids and had a conserved EIN3 domain. Realtime Quantitative PCR (qRTPCR) showed that the relative expression of GhEIL3 after the treatment of Fusarium oxysporum (Fov.) was significantly higher than the control group and reached its maximum after induction for 1 h. The results demonstrated that GhEIL3 may participate in the defensive reaction of the Fov. in cotton.