Current studies of fungal bioluminescent systems, including the purification of luciferase, require large quantities of the synthetic substrate luciferin available. The existing synthetic method for the luciferin produces low yield of the target molecule. In the current work, the synthesis of fungal luciferin was scaled up to gram quantities and an effective method for its final purification was found. The total yield in comparison with the previous method was increased by 3.5 times; the overall cost of synthesis fell by 3 times.