In this study, a fractionation and valorisation scheme for vine shoots is proposed for biofuel and lignin production. This agricultural waste was fractionated by acid/organosolv sequential pretreatment. In the first step, acid pretreatment was optimised at 150ºC and 1.2% H2SO4 to release hemicellulosic sugars, of which 76% could be recovered. This sugar stream was co-fermented by E. coli with an ethanol yield higher than 98% after detoxification with resins or NH4OH. The solid obtained under optimal acid pretreatment conditions was delignified by organosolv treatment, and a delignification rate of 43% was reached at 180ºC. This substrate with 83% enzymatic digestibility was bio-converted into ethanol by simultaneous saccharification and fermentation, with a yield of 76%. Additionally, lignin was recovered from the organosolv liquor, aiming for the full valorisation of the biomass, which showed a syringyl/guaiacyl ratio of 0.92 by nuclear magnetic resonance, complying with the data provided for Fourier transform infrared spectroscopy and confirming the aromaticity of this fraction for further valorisation.