Peritrichously flagellated bacteria such as Escherichia coli (E. coli) perform chemotaxis by a biased random walk toward various chemicals, which was driven by the bacterial flagellar motor. Fructose, a typical monosaccharide that can attract E. coli. However, little is known about the chemotaxis and motility response of E. coli towards fructose. Here, we characterized the chemotaxis behavior of E. coli to different concentrations of fructose from 0 mM to 50 mM by using microfluidics and bead assay. We observed the wild-type cells responded to the stimulus of fructose, which suggested fructose is an attractant to E. coli, while the cells defective in chemotaxis could not sense the stimulus of fructose. The motility of wild-type cells was reduced in various concentrations of fructose, which helped the aggregation of cells near surfaces, in contrast with the result that the fructose showed no effect on the motility of the cells defective in chemotaxis. Similar phenomena are expected to be found in the effect of other monosaccharides to E. coli.