Molybdenum oxide nanoparticles (NPMo) is a novel clean molybdenum source fertilizer known for enhancing grain yield in agricultural crops, including soybean. However, the impact of NPMo application on soybean's nutritional quality remains unclear. To investigate, a potted plant experiment compared soybeans response to three molybdenum sources (NPMo, bulk molybdenum trioxide (BMo), and ammonium molybdate (IMo)), focusing on mineral, fatty acid, and amino acid contents. The results showed that Mo application could improve yield of soybean, as supported by the increased index of yield composition. Further study showed that Mo application increased the concentrations of macronutrients and micronutrients by improve root traits and nodule number. In addition, Mo application optimized the composition of fatty acid by improving polyunsaturated fatty acids via the increased contents of linoleic, linolenic acid and decreased oleic acid in soybean seed. The essential amino acid (EAA) and total amino acid (TAA) content of seeds in soybean-treated with IMo and BMo was higher than that in NPMo treatment, but the EAA/TAA ratio of amino acid in IMo and BMo treatment was lower than that in NPMo treatment, indicating that more bioavailability of seeds in NPMo treatment and this was verified by the higher value of essential amino acid index and biomass value in NPMo treatment. The study finally suggested that NPMo fertilizer increased yield and nutritional quality of soybean.