Ethanol and biodiesel industries stand out not only to produce biofuels, but also for the generation of residual biomass. Through anaerobic digestion (AD), it is possible to produce hydrogen (H2) and methane (CH4) from by-products of these industries. The goal of this work is to value vinasse, filter cake (FC), and glycerin, through anaerobic co-digestion (co-AD), to produce H2 and CH4 in a two-stage system. Three batch experiments on a laboratory scale were carried out: preliminary tests, to investigate the proportions of the three substrates that achieve the best biogas yields; the tests of optimum concentration of glycerin, to identify the optimum concentrations of the substrate to obtain the best biogas yields; and the Biochemical Hydrogen (BHP) and Methane (BMP) Potential, to compare anaerobic mono-digestion (mono-AD) and co-AD in terms of absolute production, yield and velocity of biogas, H2 and CH4 production,. Based on the results of BHP and BMP, an energetic and economic study was carried out to evaluate the possibility of producing and applying biogas in a real scenario in an ethanol plant. Preliminary tests indicated that high substrate concentrations (above 10 g VS L -1 ) have an inhibitory effect on biogas production, and that the balance of vinasse and FC proportions are beneficial for the process. The tests of optimum concentration of glycerin showed that concentrations above 30 g L -1 do not result in an increase in CH4 production. BHP and BMP showed that co-AD brings improvements in several operational parameters, such as yield and speed of biogas production, and pointed out advantages of using glycerin for H2 production. The co-AD of vinasse and glycerin resulted in 257,15 ml CH4 g VS -1 and 64,31 ml H2 g VS -1 . The energy evaluation indicated that, within the proposed scenario, it is possible to achieve production of 20 thousand Nm 3 of biogas per day and replace 30% of the diesel used by agricultural machinery, considering a plant with a processing capacity of 3 million tons of sugarcane per year (2,27 Nm 3 t -1