The batik industry produces wastewater with a high dye content. One of the well-developed batik industry businesses is in Sokaraja Banyumas. A potential method for processing batik waste is by utilizing indigenous mushrooms. Aspergillus sp. 3 can be used for decolorization of Indigosol Blue batik waste. Decolorization of batik waste can use immobilization techniques or attachment to the substrate. Bentonite is a type of clay mineral that has the potential as a support material for immobilization. This study aims to determine the ability of the fungus Aspergillus sp. 3 which was immobilized by bentonite with different incubation times for the decolorization of batik waste, and to find out the effective incubation time for the use of Aspergillus sp. 3 which is immobilized by bentonite against the decolorization of batik waste.This study used a Completely Randomized Design (CRD) experimental method with incubation times of 0, 6, 12, 18, 24, 30, 36 hours. The independent variable is incubation time, while the dependent variable is the ability of the fungus Aspergillus sp. 3 which is immobilized by bentonite in the decolorization of batik waste. The main parameter is the percentage of decolorization of IB batik waste, while the supporting parameters are pH, temperature, TDS, and repeated use of bentonite immobilized mycelium pellets. The percentage of decolorization data was analyzed using analysis of variance at an error rate of 5% followed by the Advanced Significant Difference test with an error rate of 5%. Incubation time of 6 hours is an effective time for Aspergillus sp. 3 immobilized bentonite for decolorizing batik IB waste was 97.43%, pH ranged from 5.40-7.30, temperature ranged from 30-31 C, TDS percentage ranged from 14.63-40.31, and repeated use of immobilized bentonite mycelium pellets up to 3X.