This study presents the effect of different factors like initial concentration of dye, carbon and nitrogen sources, enzyme mediator, cofactor and heavy metal ions in culture medium for decolorization of Methyl Violet 2B, an azo dye widely used in textile and leather industries, through Pleurotus sajor-caju spent substrate. Spent substrate of
P. sajor-caju was recorded for highest decolorization (100%) with lowest concentration (25 ppm) of Methyl violet 2B. Addition of carbon and nitrogen sources separately, except cellulose enhanced decolorization and highest was in presence of sucrose (87.75%), followed by starch (87.48%). Lower concentration (25 ppm) of glucose, starch and ammonium carbonate, while higher concentration of urea (100 ppm) supported highest decolorization. After 4 to 5 days of incubation, highest decolorization was with 0.1% Veratryl alcohol and Manganese ions added separately in culture medium, while presence of salts of lead, cadmium, cobalt and nickel (0.025%) in culture medium supported higher decolorization of 100, 94.60, 71.0 and 68.54%, respectively against 62.35% in control.  Among the ligninolytic enzymes the spent substrate exhibited appreciable activities of laccase, manganese peroxidase and lignin peroxidase.