Herein, the wool fabric was mordanted with alum, treated with microbial transglutaminase (m-TGase), and then dyed with madder. Different concentrations of alum and m-TGase were used to find out the optimum condition to achieve the best color after dyeing the wool fabrics with aqueous extract of madder. FT-IR spectroscopy and scanning electron microscopy (SEM) methods were applied to characterize the as-prepared samples. Contact angle measurements showed that the water uptake capability was increased in the case of the wool sample treated with alum and enzyme. Moreover, the samples were assessed for color strength (K/S) and color fastness. Our results showed that the optimal condition to get the highest color value was for the sample with 10% owf (of weight of fabric) alum and 5% owf m-TGase. Furthermore, it was found that there was a critical concentration for enzyme so that an increase in m-TGase amount would cause damage to the scales of fibers. The best condition of the dyeing process was discussed in this study, and also the proposed mechanism was presented. Indeed, treatment of wool with m-TGase led to a reduction in the amount of consumed alum, while investigations in color performances demonstrated the enhancement in color fastness, as well as color strength.