A novel large-effect QTL qPCB03 for protein concentration was identified, and a KASP marker linked to qPCB03 was developed and validated to enhance protein concentration by 1.23 to 1.57 percentage points in the RIL populations. Peanut is a vital source of protein for humans, playing a key role in maintaining a steady protein supply. In this study, the high-protein cultivar Zhonghua6 (29.14 ± 1.69%) was crossed with Xuhua13 (24.55 ± 1.84%) to construct a recombinant inbred line (RIL) population. The protein concentration of the RIL population exhibited significant variation, ranging from 21.05 to 30.28%. To discover genomic regions associated with protein concentration, four libraries were constructed (two parents and two extreme bulks) for bulked segregant sequencing (BSA-seq). The results revealed significant associations between protein concentration and the genomic regions on chromosomes A07, A10, B03, and B10. Through linkage analysis, nine QTLs (quantitative trait loci) for protein concentration were identified, among which the large-effect and stable QTL qPCB03 on chromosome B03 (125.92-127.94 Mb) explained 11.03-12.86% of the phenotypic variation. This QTL (qPCB03), simultaneously identified by both BSA-Seq and linkage mapping, had not been documented in prior studies. Within the ~ 2 Mb interval of qPCB03, a total of 349 genomic variants were discovered, including six single nucleotide polymorphisms (SNPs) that resulted in nonsynonymous mutations in six genes. Based on genomic variants of the target region, KASP markers were developed and validated in 147 peanut varieties exhibiting variable seed protein concentration. According to the marker-assisted selection, favorable genotypes could potentially enhance protein concentration by 1.23-1.57 percentage points in the RIL populations. The identification of stable loci and the development of markers facilitate marker-assisted breeding in peanuts, while the discovery of candidate genes lays the groundwork for the fine mapping of key genes regulating protein concentration.