The protein content in soybean is genetically controlled by multiple genes. Identification of the quantitative traits (QTL) associating with this trait will benefit for a practice use in soybean breeding. In this study, a backcross population consisting of 384 lines was derived from a cross of elite cultivar Heihe50 as recurrent parent and high protein accession ZhongYin1106 as donor parent. The protein content of parents and the offspring segregants was detected by Near-infrared spectrometer, and genotyping was conducted with SSR molecular markers. A genetic linkage map was constructed by software QTL ICIMapping4.1. Nine QTL associating with protein content were revealed, including seven QTL detecting by interval mapping (IM) and 3 QTLs detecting by inclusive composite interval mapping (ICIM). One QTL qPro-8-1, which was located in a 218.71kb interval of markers SSR_50 and SSR_51, can explain 2.26% and 7.85% phenotype variance using both IM and ICIM mapping methods, respectively. The locus qPro-8-1 is genetically independent of other QTL of protein content. Thus, this work provided elite materials and theoretical foundation in molecular breeding for high-protein soybean varieties.