In this study, the effect of Sulfur (S) on the content of kernal isoflavone in different soybean varieties was analyzed. The best Sulfur levels of different genotype soybean varieties were explored, in order to increase the content of kernal isoflavone and improve their qualities. Three soybean varieties (‘Heinong-48’, high-protein cultivar; ‘Heinong-37’, middle cultivar; and ‘Heinong-44’, high-oil cultivar) were investigated. The soybean seeds were planted in pots fertilized with S which were S1, S2, S3 and S4 (i.e., 0 g, 0.02 g, 0.04 g and 0.06 g of Sulfur) per kilogram of soil. The content of total kernal isoflavone in different soybean varieties was determined by ultraviolet spectrophotometry. The results showed that, the content of total isoflavone under the same variety of different treatment, HN37 and HN44 treatment was significantly higher than that of S2, HN48 in S3 processing under the highest content. The three varieties are suitable S is known to help improving the total isoflavone content; same deal with the different varieties of soybean total isoflavones content, under the S1-S4 treatment HN44 was significantly highest, and then HN37 and HN48. Under different treatments in different soybean varieties, the content of total isoflavone in HN44 S3 was the highest. These findings suggested that Sulfur affected the total content of isoflavone in the three soybean varieties, and appropriate amounts of S fertilization might contribute to improving the content of kernal isoflavone.