Abstract: Plant nitrogen content was a vital indicator to evaluate pasture growing situations, to estimate production and to determine optimal fertilizer application. An experiment was conducted to investigate the plant nitrogen content of annual ryegrass and the characteristics of canopy spectral reflectance under five nitrogen levels, exploring the relationship between plant canopy reflectance and nitrogen content. The nitrogen level had a significant impact on the plant nitrogen content of annual ryegrass and when nitrogen fertilizer increased from 100 to 400 kg/ha the plant nitrogen content increased accordingly. In the visible region, canopy spectral reflectance rate reduced as nitrogen level increased. There was significant negative correlation between plant nitrogen content and single-band reflectance rate and the absolute value of the correlation coefficient was greater than 0.5 (P<0.01) within 487-718 nm. The optimal model of plant nitrogen content estimation by using sensitive bands was y=4.362-0.754x₅₇₉+0.351x₇₀₀, and the optimal model of plant nitrogen content estimation based on vegetation index was y=3.026-0.670DVI_(610,487)+4.997NDVI_(700,579). A strong correlation between estimated and true value of plant nitrogen content was detected (P<0.01).