Fusarium oxysporum f. sp. momordica was used as the target pathogenic fungus to screen actinomycetes that were isolated from rhizosphere soil of Momordica charantia by confrontation culture and antifungal tests of fermentation filtrate. The candidate strain 0250 had broad antifungal activity. According to cultural characteristics, physiological and biochemical properties, as well as average nucleotide identity analysis of the strains with similar homology, the strain 0250 was identified as Streptomyces rhizosphaericus . Its effects on growth promotion and control of bitter gourd wilt were evaluated in both greenhouse and field. The results showed that the plate inhibition percentage of S. rhizosphaericus strain 0250 against F. oxysporum f. sp. momordica was 69.2%, while the plate inhibition percentage against 17 plant pathogenic fungi reached 64.3%-85.6%. The suspension treatment of the strain could promote the growth and development of roots and stems and improve production of bitter gourd in pots and field. The control efficacy of Fusarium wilt of bitter gourd was 66.9% and 61.5%, respectively. When soils were treated with the strain 0250 suspension in advance and inoculated with the fungal pathogen, the inhibition percentage on the soil F. oxysporum reached 62.1%. The activity of phenylalanine ammonia-lyase, peroxidase and β-1,3-glucanase as well as root activity were significantly improved in bitter gourd seedlings. In summary, strain 0250 is an actinomycetes resource with biocontrol potential to Fusarium wilt of bitter gourd.