Biofuels are essential for transitioning to a sustainable society. This switch can be achieved by introducing novel feedstocks and technologies for efficient and economically feasible biofuel production. Second-generation biofuels are particularly advantageous, as they are produced from nonedible lignocellulosic biomass derived primarily from agricultural byproducts. Ciona intestinalis, a marine filter feeder, is cultivated to produce fish feed from the invertebrate’s inner tissue body. This process generates also vast amounts of a renewable side stream, namely the tunicate’s external cellulose-rich tunic. The aim of the present study was to evaluate the potential of the C. intestinalis tunic as a novel feedstock for bioethanol production. For this purpose, organosolv fractionation of the tunic was optimized to increase cellulose content. Enzymatic saccharification of the pretreated biomass was assessed to identify the most promising materials, which were subsequently utilized as carbon source in fermentation trials. Under optimal conditions, a titer of 38.7 g/L of ethanol, with a yield of 78.3% of the maximum theoretical, was achieved. To the best of our knowledge, this is the first report whereby organosolv pretreated tunic biomass is valorized toward bioethanol production; the current work paves the way for incorporating tunicates in bioconversion processes for the generation of biofuels and other biobased chemicals.