【Objective】 The objective of the present study was to investigate the effect of different application methods of nitrogen （15N） fertilizer on some physiological and biochemical parameters in sugarcane. 【Method】 The plants of sugarcane variety ROC 22 were cultured in soil-pot culture. The total amount of nitrogen fertilizer （5 g/pot） was kept as basic fertilizer treatment except the T1， where no fertilizer has been given to the plants. The other three treatments were: 5 g/pot at stage（T2）， 2.5 g/pot basic fertilizer+2.5 g/pot tillering fertilizer（T3）， 1.5 g/pot basic fertilizer +1.5 g/pot tillering fertilizer+2.0 g/pot elongation fertilizer（T4）. The chlorophyll and soluble protein contents， nitrate reductase activity and root activity were investigated at different stages. 【Result】 The analyzed parameters differed significantly in plants treated with different treatments. In T2， all the parameters increased significantly at seedling stage， while a decrease in all was observed at tillering and elongation stage. In T4， all the parameters were found to be significantly higher at both， the tillering and the elongation stages. 【Conclusion】 Applying nitrogen fertilizer in three splits， each at seedling， tillering and elongating stages significantly improved the chlorophyll and soluble protein content， nitrate reductase activity and root activity， and maintained the growth and development of sugarcane plants.