Abstract: Nitrogen fertilizer is the most important nutrient element for plant growth, and the amount of nitrogen applied is also very important. In order to study the effect of nitrogen fertilizer application rate on oil content and gene expression level of rapeseed, Ningyou 22 of Brassica napus L. was used as material to determine the oil content of Ningyou 22 seeds and pod peel at three different nitrogen fertilizer levels: 0 kg/hm2, 320 kg/hm2 and 640 kg/hm2. Meanwhile, G3PDH and PK genes were detected at different nitrogen fertilizer levels. The expression level in different tissues. The results showed that with the increase of nitrogen application, the oil content of Ningyou 22 decreased, but not significantly at 0 kg/hm2 and 320 kg/hm2 levels, and significantly decreased at 640 kg/hm2 levels. The expression of G3PDH and PK genes increased with the increase of nitrogen application. The ratio of G3PDH and PK gene expression in seed to pod and 15 days after flowering were also analyzed. The results showed that the ratio of G3PDH gene expression in seed to pod and 15 days after flowering was the highest at 640 kg/hm2 level. The ratio reached the highest at 640 kg/hm2 level, and reached the highest at 0 kg/hm2 level after 30 days of flowering and 15 days of heeling.