Abstract: An nutrient solution experiment with seven nitrogen application levels, and measured the SPAD value of 17 sites in different cotton leaf was conducted to investigate the correlation between nitrogen nutrition status of cotton plant and leaf SPAD value under different nitrogen application. The main aim was to determine the most appropriate measurement sites of leaf SPAD value. The results showed that, the leaf of the reverse 4th from the apex of the main stem was the best measuring leaf because of there was significant correlation between the nitrogen nutrition and SPAD values of the 4th leaf in all leaves. The correlation between the nitrogen nutrition and SPAD values was also different in different determine sites of inverse fourth leaf, which S3 (tip position tip leaf) showed the best significant correlation between SPAD and N concentration of cotton shoot. The results suggested that, the location of near tip of leaf in inverse fourth leaf was the most appropriate site to estimate the nitrogen nutrition status of cotton with SPAD value.