One of the compounds generally found in the residues of the coffee and tea industries is caffeine, which in high concentration is toxic to various organisms, making it necessary to find an adequate treatment for these residues. Biotechnological treatments using enzymes can be an alternative to valorize and detoxify these residues. However, mixtures of substrates have not been evaluated to improve production. Therefore, the present investigation aimed to study the effect of different proportions of sorghum-coffee pulp mixtures as a substrate in solid-state fermentation with the fungus Rhizopus oryzae (MUCL 28168) for the production of n-demethylases. To evaluate the synergistic and antagonistic effects of coffee pulp and sorghum mixtures on n-demethylase enzyme production, a simplex-centroid design, using four levels: 1 (100%), 1/4 (25%), 1/2 (50%), 3/4 (75%). Results obtained were favorable, achieving a caffeine demethylase activity of 18.762 U/g, and reducing the caffeine content in the coffee pulp.