Nitrogen (N) is vital important for terrestrial ecosystems, which can increase plant growth and aboveground net primary productivity (ANPP) through either enrichment artificial nitrogen application or atmospheric nitrogen deposition. Atmospheric nitrogen deposition affects the variation and evolution of plant functional traits, which in turn affects individual plant and ecosystem functions. However, the frequency of natural atmospheric nitrogen deposition is significantly higher than that of artificial nitrogen addition. Most of the previous studies often used one-time nitrogen application in the growing season, whether the frequency of nitrogen addition alters the response of plant functional traits and ecosystem ANPP still unclear. Studying the differences in the response of plant functional traits in grassland ecosystems under different nitrogen addition modes is essential to more accurately assess the adaptability of plants to environmental changes. Based on the long-term nitrogen deposition simulation experimental platform in the grassland of the Inner Mongolia, the frequency of nitrogen addition experiment was established in September 2010, with six randomly set blocks (replicates). The field experiment contained a control (without N addition) and once per year nitrogen addition, twice per year nitrogen and once per month nitrogen addition, and the effect of nitrogen addition frequency on the chlorophyll content of the dominant species of Leymus chinensis and Agropyron cristatum was studied. The experimental results showed that the cholorophyll content of L. chinensis and A. cristatum were significantly enhanced by nitrogen addition. The chlorophyll content of L. chinensis increased more under the once per year nitrogen addition (15.21%), while once per month nitrogen addition had the greatest impact on the chlorophyll content of A. cristatum (increase 14.74%). Meanwhile, nitrogen addition, especially once per year nitrogen application significantly increased soil ammonium nitrogen, nitrate nitrogen and inorganic nitrogen content, and significantly reduced soil pH, while the soil water content (SWC) was similar among the frequencies of nitrogen addition. The results showed that the chlorophyll content of L. chinensis had more obvious response to low-frequency nitrogen addition, while the high-frequency nitrogen addition played an important role in increasing the chlorophyll content of A. cristatum. and there were obvious differences in nutrient uptake strategies between these two species. This study indicates that low-frequency nitrogen addition may overestimate the effect of nitrogen deposition on chlorophyll content in L. chinensis, while underestimate the effect on A. cristatum, which is of great significance for accurately predicting the variation of plant leaves functional traits to atmospheric nitrogen deposition, and will be helpful to apply to models of plant functional traits predicting ecosystem functions and processes in response to future global changes.