Lucerne (Medicago sativa) has many qualities associated with sustainable agriculture such as nitrogen fixation and high biomass yield. Therefore, there is interest in whether lucerne is a suitable biomass substrate for bioethanol production, and if hydrothermal pretreatment (HTT) of lucerne improves enzymatic convertibility, providing sufficient enzymatic conversion of carbohydrate to simple sugars for ethanol production. The HTT process was optimised for lucerne hay, and the pretreated biomass was assessed by carbohydrate analysis, inhibitor characterisation of liquid phases, and by simultaneous saccharification and fermentation (SSF) of the whole slurry with Cellubrix enzymes and Saccharomyces cerevisiae yeast. The optimal HTT conditions were 205°C for 5 minutes, resulting in pentose recovery of 81%, and an enzymatic convertibility of glucan to monomeric glucose of 74%, facilitating a conversion of 6.2% w/w of untreated material into bioethanol in SSF, which is equivalent to 1,100 litre ethanol per hectare per year.