Soybean provides essential protein and amino acids for humans and animals, while sulfur-containing amino acids (SAA), including methionine (Met) and cysteine (Cys), are very limited. In this study, we constructed a high-density bin-map with 3420 bin markers using 676 857 SNPs of a recombinant-inbred line (RIL) population derived from a cross between Kefeng no. 1 and Nannong 1138-2. Quantitative trait loci (QTL) mapping was performed for Cys, Met, SAA, and the protein content using this high-density bin-map. Twenty-five QTLs linked to these four traits were identified, and four genomic regions located on chromosomes (Chr) 07, 08, 15, and 20 were overlapped by multiple QTLs. Among them, bin 115-124 located on Chr 15 was associated with all four traits and was a novel locus with a high LOD value. These findings will provide a basis for nutritional quality improvement using marker-assisted selection breeding and clarify the genetic mechanisms of SAA and protein in soybean.