This study is based on the 10 year old Cabernet ‘Gernischet’, which was treated with 300 kg·hm^-2 urea at the germination stage (S1), new shoot vigorous growth stage (S2), flowering stage (S3), the first expansion stage of fruit (S4), secondary shoot vigorous growth stage (S5) and the second expansion stage of fruit (S6). No nitrogen fertilizer was used as the control (CK). The indicators in leaves were analyzed at 5 days before flowering (DBF5), 25 days after flowering (DAF25), 55 days after flowering (DAF55), and 85 days after flowering (DAF85), for clarifying the regulation and influence of nitrogen application period on nitrogen metabolism in grape leaves. The results showed that: (1) S1 and S2 treatments significantly increased the total nitrogen and soluble protein contents in leaves at DAF 25. (2) The activities of NR and GS in S3 and S4 treatments were significantly higher than those in other treatments at DAF 85. At DAF 25, the GOGAT activities of S1 and S2 were 0.29 and 0.33 μmol·g^-1·min^-1, respectively. The GDH activities of S3 and S4 significantly higher than those of control and other fertilization treatments, which were 225.0 and 227.3 U·g^-1, respectively. (3) Quantitative analysis showed that the expression level of VvNR was higher than that of control at different stages, and was 3.4 times and 2.7 times higher than that of control in S3 at DAF25 and DAF85, respectively. The expression levels of VvGS in S3 and S4 were the highest at DAF55 and DAF85, respectively. The expression level of VvGOGAT in S3 and VvGDH in S4 were significantly higher than that of other treatments at DAF55 and DAF85. In addition, the expression level of VvGDH in S3 at DAF55 and DAF85 was second only to S4. Correlation analysis showed that there was a linear relationship among nitrogen content, nitrogen metabolism enzyme activities and the expression levels of regulatory genes. Therefore, nitrogen could induce the response of leaf nitrogen metabolism genes, thereby regulating nitrogen metabolism in leaves. S3 and S4 treatments were higher in the activities of nitrogen metabolizing enzymes and the corresponding gene relative expression levels in different periods, and were more conducive to the transformation and metabolism of nitrogen in the leaves. This study could provide some support for the scientific application of nitrogen fertilizer.