Direct yellow 4 degradation was investigated in the presence of various mediators (p-coumaric acid, 1, hydroxybenzotriazole (HOBT), syringaldehyde, vanillin, syringic acid, veratryl alcohol, and pyrocatechol) at pre-optimized conditions of process variables, i.e., pH 5.0, temperature 50ºC, enzyme dose 24 U/mL, and 0.25 mM H₂O₂ concentration. Citrus limon peroxidase (CL-POD) was used for degradation of DY4 dye. In the absence of mediators, the DY4 degradation was 60%, whereas mediators enhanced the POD biodegradation efficiency up to 87%. Among mediators investigated, syringaldehyde showed promising efficiency. We investigated yringaldehyde concentrations in the range of 0.0125-0.5 mM, and 0.025 mM was optimum for maximum dye degradation, which revealed that mediators could be used to enhance the biodegradation of dyes.