In order to identify the seasonal variations and dominant driving factors of NO3-N in rivers, investigations of five consecutive years were conducted in seven rivers of the Miyun Reservoir Watershed. Significant seasonal variation of NO3-N in rivers was separately found in the dormant season (non-growing season) and the growing season. Furtherly, the V-shaped, W-shaped, and indistinct seasonal patterns of NO3-N accounted for 53.0%, 38.7%, and 8.3%, respectively. They were remarkably affected by stream flow, and their significant quadratic function was discovered. The annual maxima and minima of NO3-N corresponded to medium flow in the dormant season and low flow or flood in the growing season, respectively. On one hand, flood mainly played a role in the diluent for the Chao River with high NO3-N, and on the other hand, it acted as a nitrogen source for the Bai River with low NO3-N. The NO3-N was closely correlated with human activities, and this correlation had obvious seasonal change trend. In the dormant season, significant and mostly extremely significant high correlation coefficient (R) values were determined, while partly non-significant with low R values were found in July, August, September, and October. Increasing seasonal variation index of NO3-N from upstream to downstream was found that was gentle for large rivers and sharp for small tributaries. The seasonality of NO3-N was more affected by natural factors, especially flood, than human factors.