Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei β-fructosidase was overexpressed as truncated cytosolic protein (tfosEp) in Escherichia coli. Purified tfosEp was thermostable (10–50 °C) with a pH optimum of 5; it showed highest affinity for bacterial levan (β[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (β[2-1] linkages) and sucrose (Km values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with tfosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer’s yeast and GJ + tfosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[±4.3] and 28.2[±2.7] mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of β-fructosidase from L. paracasei.