Recently, natural dyes have a widening scope in various traditional and advanced applications due to their eco-friendly environment. However, improved dyeability of natural dyes still remains a challenging task. This research was aimed to achieve multi-objective wool with improved dyeability using bio-nano-mordant composed of m-Trans-glutaminase, m-TGase, and bentonite nanoclay. Wool fiber was treated through sonochemical method using different concentrations of m-TGase and bentonite. The surface morphology of wool fabric samples was examined by field emission-scanning electron microscopy (FESEM), and Fourier transform Infrared Radiation (FTIR). Further, wool samples treated at different conditions were applied to madder for dyeability examination. The optimum conditions of color coordinates, color strength, K/S, and washing fastness of madder on treated wool fabric with m-TGase and bentonite, were also examined. The results revealed well-made interactions among m-TGase, bentonite, and wool fibers. In addition, surface morphology was strongly influenced by variations in enzyme concentrations so that extra addition of m-TGase lead to clear damage scales or less cuticle surface in SEM images. Moreover, the results showed that the value of K/S for treated wool samples was better than untreated samples. Indeed, amongst all, 5% concentrations of bio-nano-mordant for m-TGase and bentonite have the most constructive K/S values. Similarly, results of
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E and antibacterial investigations also confirmed its superiority.