Abstract: 【Objective】 The shortage of healthy seedlings has been an important factor limiting the large scale of afforestation of Santalum album L.. A fertilization experiment was conducted to study the proper nitrogen application rate for healthy growth and high N efficiency in Santalum album L. seedlings. We assess the growth characteristics, photosynthesis performance, nutrient accumulation and the nitrogen use efficiency, aiming to provide a reference for fertilization technique in mass planting Santalum album L. seedlings in greenhouse. 【Methods】 A pot culture experiment was conducted inside greenhouse. Nitrogen application rate was designed in exponential folds: 0, 50, 100, 150, 200, 300 and 400 mg per seedling. The nitrogenous fertilizer was divided into12 parts and applied at an interval of 10 days. The water content in the culture substrate was kept at 70% of field capacity by weighing method. The height, ground diameter, biomass, root-shoot ratio, chlorophyll content, chlorophyll fluorescence, nitrogen absorption,apparent N recovery efficiency and nitrogen agronomy efficiency of the seedlings were measured. 【Results】 1) The height, ground diameter and biomass were increased until the nitrogen application rate was 400 mg/seedling, with the highest value of 24.37 cm, 2.87 mm and 1.17 g respectively, but the root-shoot ratio decreased to the lowest value of 0.36.2) Similarly, the chlorophyll content(Chl a +b), Fv/Fm, ETR were increased until the nitrogen application rate of 400 mg/seedling, with the highest values of 1.40 mg/g(FW), 0.727 and 27 respectively, whereas the highest Fm(0.568) and yield(0.614 g) were achieved at nitrogen application rate of 300 mg/seedling. 3) The N uptake was kept increase, but the nitrogen agronomy efficiency started to decrease when the nitrogen application rate was over 200 mg/seedling. 【Conclusions】 Nitrogen application rate has significant effect on the growth of Santalum album L. seedling. Considering the growth characteristic, photosynthesis performance, nutrient accumulation and nitrogen use efficiency, supply of nitrogen application rate 300 mg/seedling not only exhibits high growth and biomass effects, but also improves the agronomy efficiency of nitrogen fertilizer, proving to be a proper nitrogen application rate for Santalum album L. seedlings in greenhouse in this study