Abstract: This study was to oriented screen antagonistic Bacillus against Fusarium oxysporium f. sp. cucumarinum Owen and analyze the character of its antifungal agents. Heat treatment and morphological observation combining flat confrontation culture method was used to oriented screen antagonistic Bacillus against Fusarium oxysporium. Sixty three isolates of spore-producing bacterial strains were isolated from the rhizosphere soil of protected continuous cropping cucumber. Among them, two strains, named as j-50 and j-28, were found to have high antifungi activities against Fusarium oxysporium. Especially, strain j-28 had an inhibition zone of 11.0 mm against Fusarium oxysporium. Furthermore, strain j-28 also had high antagonistic effects on other plant pathogenic fungi. The strain j-28 was identified as Bacillus subtilis by physiolofical characteristics, biochemical, morphological, as well as the 16S rDNA sequence analysis. This study showed that antifungal activities of strain j-28 were significantly affected by heat and acid-base treatment, which may suggested that the antifungal agents of strain j-28 may be protein. Bacillus j-28 with high antifungal activities and its special characters can be a potential biological control source of Fusarium oxysporium.