Sugar accumulation and content are major characteristics of watermelon fruit quality. The sweet watermelon inbred 97103 and the non-sweet watermelon accession PI296341-FR were used to study the enzymology and transcriptome dynamics of sugar metabolism and accumulation during watermelon fruit development. We found that differences of the sugar metabolism enzyme activities lead to the different photosynthate assignment. Acid a-galactosidase and insoluble invertase activities may play an important role in oligosaccharides hydrolyzation and sucrose accumulation in 97103. Soluble acid invertase may contribute to the decomposition from sucrose to glucose and fructose. The high activities of sucrose synthase and sucrose phosphate synthase are necessary for accumulating sugars in sweet watermelon fruits. The digital expression profiling of both flesh and mesocarp at four critical stages of fruit development of 97103 further confirmed these enzyme activities in transcriptome level. These results indicate that the corporate action of photosynthate phloem unloading, partitioning and metabolism determine the sugar content in sweet watermelon and non-sweet watermelon.