Soybean (Glycine max) is the main oilseed crop that provides vegetable oil for human nutrition. The main objective of its breeding research is to increase the total oil content. In the Kennedy pathway, Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme that converts diacylglycerol (DAG) to triacylglycerol (TAG). Here, the AhDGAT3 gene was cloned from peanut and overexpressed in the wild-type (WT) Arabidopsis. The total fatty acid content in T₃AhDGAT3 transgenic Arabidopsis seeds was 1.1 times higher on average than that of the WT. Therefore, AhDGAT3 was transferred into the WT (JACK), and four T₃ transgenic soybean lines were obtained, which proved to be positive using molecular biological detection. Specific T-DNA insertion region location information was also obtained via genome re-sequencing. The results of high-performance gas chromatography showed that the contents of oleic acid (18:1) composition and total fatty acids in transgenic soybean plants were significantly higher than that of the WT. However, linoleic acid (18:2) was much lower compared to the WT. The agronomic trait survey showed that the quantitative and yield traits of AhDGAT3 transgenic soybean were better than those of the WT. These results suggest that fatty acids in transgenic soybeans, especially oleic acid and total fatty acid, are enhanced by the over-expression of AhDGAT3.