Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation ofStreptomyces fradiae; however, it is necessary to modifyS. fradiaestrains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifyingS. fradiaestrains that produce increased yields of tylosin. Additionally, we constructed mutant libraries ofS. fradiaevia ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identifiedS. fradiaemutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 μg/ml) and UN-C137 (6889.72 ± 70.25 μg/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 μg/ml). These mutant strains will form the basis for further strain breeding in tylosin production.