[Background] Cucumber Fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum, which seriously restricts the development of cucumber industry. [Objective] Screening and identification antagonistic actinomycete strains with strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum was isolated from saline-alkali soil in Hexi corridor. We identified the antagonistic actinomycete strain and optimized the fermentation conditions. [Methods] The method of dilution plate coating was applied to isolate actinomycetes; The methods of growth plate-confrontation and speed rate were applied to screen antagonistic actinomycetes; The morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence of strain 16-3-10 were analyzed the taxonomic status. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment. [Results] The result of identification revealed that strain 16-3-10 can be classified as Streptomyces sp., the optimum culture conditions of strain 16-3-10 were with a medium was as follows: millet 10.0 g/L, lactose 20.0 g/L, peptone 1.0 g/L, NaCl 5.0 g/L, CaCO3 6.0 g/L at 28 °C, liquid volume 50 mL in 250 mL flask, initial pH of 10.0 for 3 d, and inhibition rate of strain 16-3-10 to pathogen reached to 82.50% under this condition. [Conclusion] The result indicates that 16-3-10, strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum, might act as a potential candidate for the application.