Enzymes that participate in reaction cascades have been shown to assemble into metabolons, in the presence of the first enzyme's substrate. However, the mechanism of metabolon formation is still an open question. We applied microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cycle: hexokinase, phosphoglucose isomerase, phosphofructokinase, and aldolase and showed that each enzyme independently follows its own specific substrate gradient, which is produced by the preceding enzymatic reaction. The extent of enzyme migration is proportional to the time the enzyme is exposed to the substrate gradient. Significantly, the chemotactic migration of enzymes is fairly rapid even under conditions that mimic cytosolic crowding. The observed rate was very similar to the reported rate of enzyme diffusion in living cells. Thus, chemotaxis may be a basis for the organization of metabolic networks in the cytosol of the cell.