Soybean is a cereal crop with high protein and oil content which serves as the main source of plant-based protein and oil for human consumption. The symbiotic relationship between legumes and rhizobia contributes significantly to soybean yield and quality, but the underlying molecular mechanisms remain poorly understood, hindering efforts to improve soybean productivity. In this study, we conducted a transcriptome analysis and identified 22 differentially expressed genes (DEGs) from nodule-related quantitative trait loci (QTL) located in chromosomes 12 and 19. Subsequently, we performed functional characterisation and haplotype analysis to identify key candidate genes among the 22 DEGs that are responsive to nitrate. Our findings identifiedGmTCP(TEOSINTE-BRANCHED1/CYCLOIDEA/PCF) andGmNLP(NIN-LIKE PROTEIN) as the key candidate genes that regulate the soybean nodule phenotype in response to nitrogen concentration. We conducted homologous gene mutant analysis inArabidopsis thaliana, which revealed that the homologous genes ofGmTCPandGmNLPplay a vital role in regulating root development in response to nitrogen concentration. We further performed overexpression and gene knockout ofGmTCPandGmNLPthrough hairy root transformation in soybeans and analysed the effects ofGmTCPandGmNLPon nodulation under different nitrogen concentrations using transgenic lines. OverexpressingGmTCPandGmNLPresulted in significant differences in soybean hairy root nodulation phenotypes, such as nodule number (NN) and nodule dry weight (NDW), under varying nitrate conditions. Our results demonstrate thatGmTCPandGmNLPare involved in regulating soybean nodulation in response to nitrogen concentration, providing new insights into the mechanism of soybean symbiosis establishment underlying different nitrogen concentrations.