[Background] Watermelon Fusarium wilt is a soil-borne disease caused by Fusarium oxysporum f. sp. niveum, and one of the most serious diseases in watermelon production. Identification of antagonistic microorganisms is necessary to develop biocontrol agents. [Objective] To isolate and identify effective biocontrol strains against watermelon Fusarium wilt, and investigate the inhibitory effect, ultimately provide candidates for developing biocontrol agents. [Methods] We isolated bacteria from the rhizosphere soil of watermelon. Antagonistic bacteria were screened by plate confrontation and greenhouse bioassay method. Morphological observation and gyrB gene sequence homology analysis were performed to characterize the strains isolated. The antagonistic factors (cellulase, protease, chitinase, β-1,3-glucanase) of antagonistic bacteria were determined by differential medium, and the effects of antagonistic bacteria fermentation liquid on the spore germination and hypha growth of Fusarium oxysporum f. sp. niveum were determined. Finally, the control effect of antagonistic bacteria on watermelon Fusarium wilt was determined in the field. [Results] An antagonistic strain SFJ11 against watermelon wilt was selected by means of plate confrontation and greenhouse assay. The strain was identified as Bacillus amylolyticus by morphological identification and homology analysis of gyrB gene sequence which could secrete protease and cellulase. The 20% fermentation broth of antagonistic bacterium SFJ11 could almost completely inhibit the growth of mycelia, and inhibit the germination of 95.44% of spores of Fusarium oxysporum f. sp. niveum. The control effect of SFJ11 on watermelon Fusarium wilt was 78% in field experiment. [Conclusion] Strain SFJ11 had a notably biological control effect on watermelon Fusarium wilt, and it would be a potential biocontrol agent.