Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl‐ and monoaminoethyl‐N‐aminoethyl (MANA)‐Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein–supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase‐MANA‐Sepharose CL 4B immobilized derivative is better than laccase‐glyoxyl‐Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6‐dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase‐MANA‐Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

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