The invention relates to a production method capable of increasing yield of neomycin. According to the production method, one or several of additives which are lysine, ferrous lactate, tyrosine, methionine, hydrolysis casein, vitamin B12, fructose, glucosamine, folic acid, riboflavin and tryptophan are added to a conventional fermentation medium comprising peanut meal, corn starch, rice flour, yeast powder, glucose, peptone, ammonium sulfate, sodium chloride, light calcium carbonate and KH2PO4-containign streptomyces fradiae. The production method has the advantages that the yield of the neomycin is increased by above 5-20%, the cost is reduced, and the fermentation efficiency is high. The neomycin is used for treating human diseases, widely used in animal husbandries and used as antibiotics.