BLAST searches of cotton EST database were performed using the AP2/ERF domain of the ERF-B3 subgroup transcription factors from Arabidopsis thaliana as a query sequence.Through the method of in silico cloning and RT-PCR,GhB301,an ERF-B3 subgroup transcription factor related to resistant Fusarium oxysporum f.sp.vasinfectum(Fov) was cloned from the roots of ‘Zhongmiansuo 12’ which was a high resistant cultivar to the infection of Fov.Sequence analysis indicated that the full length cDNA of GhB301 was 593 bp,with 384 bp ORF encoding 127 amino acids and a conserved AP2/ERF domain.qRT-PCR analysis indicated that,GhB301 gene highly expressed in roots and resistant cultivar after the treatment of Fov.The expression level of GhB301 also changed under the treatment of ET,SA,drought,low temperature and high salinity.The results suggested that GhB301 gene might participate in the response of plants to the pathogenic-bacteria,hormone and abiotic stresses.