Sugarcane (Saccharum spp.) is a dynamic C4 polyploidy grass used as a major source of sucrose and an alternative for ethanol, food, and energy. Despite growing scientific interest, various sucrose metabolism regulatory aspects have been limited. Biochemical and gene expression studies were conducted on developmental stages, 240-420 days of planting (DAP) in mature leaves of three high and three low sucrose sugarcane cultivars. Sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were found to be remarkably higher at 240-360 DAP but decrease at 420 DAP. Twofold increases of SS activity was estimated at 240-360 DAP while SPS activity trend was found to be lower than the SS activity. In comparing SS and SPS activities with the brix of respective DAP, results show that these activities are significant and positively correlated with 'r' values of 0.69 and 0.68 for SS and SPS, respectively. However, the soluble acid invertase (SAI) and neutral invertase (NI) activities were found to decrease significantly with the maturity of cultivars, negatively correlating with brix at 'r' values 0.83 and 0.89 for SAI and NI, respectively. The antioxidant enzyme activity was modulated similar to the invertases activity. Of the six genes, ESAS 11 and 23 associated with sucrose accumulation and ESTS 34 and 41 associated with sugar transport in sugarcane were differentially expressed among the selected high and low sugarcane cultivars. Hence, these findings reinforce the selection of diverse sugarcane cultivars for gene expression studies targeting to quantitative traits and candidate marker determination.