Abstract: Traditional coloring process of Changsha stinky tofu shows some disadvantages such as oxidation-induced fading and accelerated corrosion of stainless-steel equipment. To explore an alternative coloring process, melanin synthesized through the enzymatic method was used for coloring stinky tofu. The pH regulators for melanin solution preparation were screened. The impacts of pH levels and the addition of auxiliary factors on coloring effects were investigated. Color stability studies and sensory evaluations were also conducted on raw stinky tofu and fried stinky tofu while the coloring mechanism further explored using molecular docking simulations and particle size determination. The results showed that sodium carbonate was demonstrated better suitability compared with other tested pH regulators for preparing melanin solution, and the coloring effect was significantly enhanced with increasing pH of the melanin solution, reaching a dark black coloring effect at pH10.0. When 25 g/L maltitol or sorbitol was added into the melanin solution as auxiliary factor, resulted in a dark black coloring effect at pH8.5. The melanin-colored raw stinky tofu exhibited color stability in air, clear water, and stinky tofu brine, whereas the traditional raw stinky tofu faded to gray-white after 8 h in air. Melanin-colored stinky tofu retained its normal block shape, color, and flavor after frying. Furthermore, melanin formed stable complexes with 7S and 11S proteins in tofu mainly through hydrogen bonding and cation-π interactions. Auxiliary factors, sorbitol, maltitol, and Tween 80 significantly reduced melanin particle size in solution, while the particle size decreased with increasing pH. These findings provide new insights and a theoretical basis for alternative to traditional coloring process of Changsha stinky tofu that using ferrous sulfate as the primary coloring agent.