n-Butanol is a renewable resource with a wide range of applications. Its physicochemical properties make it a potential substitute for gasoline. Saccharomyces cerevisiae can produce n-butanol via amino acid catabolic pathways, but the use of pure amino acids is economically unfeasible for large-scale production. The aim of this study was to optimize the production of n-butanol by S. cerevisiae from protein-rich agro-industrial by-products (sunflower and poultry offal meals). By-products were characterized according to their total protein and free amino acid contents and subjected to enzymatic hydrolysis. Protein hydrolysates were used as nitrogen sources for the production of n-butanol by S. cerevisiae, but only poultry offal meal hydrolysate (POMH) afforded detectable levels of n-butanol. Under optimized conditions (carbon/nitrogen ratio of 2 and working volume of 60%), 59.94 mg/L of n-butanol was produced using POMH and glucose as substrates. The low-cost agro-industrial by-product showed great potential to be used in the production of n-butanol by S. cerevisiae. Other protein-rich residues may also find application in biofuel production by yeasts.