The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is one of the most destructive plant-parasitic nematodes in soybean production worldwide. The use of resistant soybean is the most effective alternative for its management. However, SCN-resistant soybean cultivars with increased yield and favorable agronomic traits remain limited in the market. Here, we developed a new SCN-resistant soybean cultivar Nongqing 28 from the cross the female parent cultivar ‘An 02-318’ × a male parent line F2 (Hei 99-980 × America Xiaoheidou). Resistance evaluation suggested that Nongqing 28 displayed stable resistance to SCN race 3 in pot assays and the 4-year field experiments, including inhibition of SCN development and reduction in female and cyst numbers. The average yield of Nongqing 28 were 2,593 kg/ha and 2,660 kg/ha in the 2-year regional trails and the 1-year production trials, with yield increase of 6.2% and 8.1% compared with the local cultivar Nengfeng 18, respectively. The average seed-fat contents in Nongqing 28 reached 21.26%. Additionally, RNA-seq analysis revealed that the resistance of Nongqing 28 to SCN infection is involved in pathogen perception and defense activation, such as reactive oxygen species burst, calcium-mediated defense signaling, hormonal signaling, the MAPK signaling cascade, and phenylpropanoid biosynthesis. In summary, this study provides a detailed characterization of a novel SCN-resistant soybean cultivar with high-oil and yield potential.