AbstractOptogenetics is an attractive synthetic biology tool for controlling the metabolic flux distribution. Here, we demonstrated optogenetic flux ratio control of glycolytic pathways consisting of the Embden–Meyerhof–Parnas (EMP), pentose phosphate (PP), and Entner–Doudoroff (ED) pathways by illuminating multicolor lights using blue light‐responsive EL222 and green/red light‐responsive CcaSR in Escherichia coli. EL222 forms a dimer and binds to a particular DNA sequence under blue light; therefore, target gene expression can be reduced or induced by inserting a recognition sequence into its promoter regions. First, a flux ratio between the PP and ED pathways was controlled by blue light using EL222. After blocking the EMP pathway, the EL222‐recognition sequence was inserted between the −35 and −10 regions of gnd to repress the PP flux and was also inserted upstream of the −35 region of edd to induce ED flux. After adjusting light intensity, the PP:ED flux ratios were 60:39% and 29:70% under dark and blue light conditions, respectively. Finally, a CcaSR‐based pgi expression system was implemented to control the flux ratio between the EMP and PP + ED pathways by illuminating green/red light. The EMP:PP:ED flux ratios were 80:9:11%, 14:35:51%, and 33:5:62% under green, red, and red and blue light, respectively.