Abstract: A 2-year (2008 and 2009) study was conducted to explore the effects of subtending leaf nitrogen concentration on related enzymes (Invertase, Sucrose synthase, Sucrose phosphate synthase and -1,3-glucanase) and cotton fiber strength formation in a field of Nanjing(11850E, 3202N) in middle lower reaches of Yangtze River Valley in China. Different nitrogen rates were applied to carry out different leaf nitrogen concentrations, and cotton cultivars, Dexiamian 1, Kemian 1 and NuCOTN33B were used as experiment materials. The results show that the nitrogen concentration of subtending leaves is improved with increasing of nitrogen rates, and the changes of leaf nitrogen concentration can be described as the equation: YN＝t- (YN is nitrogen concentration in the subtending leaf of cotton boll(%); t is days post anthesis(d); and are parameters). The related enzymes activities and fiber strength can be described as parabolic equations (Y=ax2+bx+c, Y is enzyme activity or fiber strength, x is leaf nitrogen concentration, a, b and c are parameters) with the increasing of the subtending leaf nitrogen concentration. During the development of cotton fiber, the subtending leaf nitrogen concentration has significant effects on related enzymes activities and fiber strength, and there are small differences among optimal leaf nitrogen concentrations of related enzymes and fiber strength. Therefore, the leaf nitrogen concentration can be used to regulate related enzymes activities to best status and to enhance the fiber strength formation. The equations of optimal leaf nitrogen concentration to days post anthesis for Dexiamian 1,Kemian 1 and NuCOTN33B are NDexiamian1=7.2841t-0.2771(R2=0.9860＊＊), NKemian1=7.1807t-0.2989(R2=0.9879＊＊) and NNuCOTN33B=7.1467t-0.2819(R2=0.9755＊＊).