Oil palm empty fruit bunch (OPEFB) fibre is widely available in Southeast Asian countries and found to have 60% (w/w) sugar components. OPEFB was pretreated using the ammonia fibre expansion (AFEX) method and characterised physically by the Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. The results show that there were significant structural changes in OPEFB after the pretreatment step, and the sugar yield after enzymatic hydrolysis using a cocktail of Cellic Ctec2® and Cellic Htec2® increased from 0.15gg(-1) of OPEFB in the raw untreated OPEFB sample to 0.53gg(-1) of OPEFB in AFEX-pretreated OPEFB (i.e. almost a fourfold increase in sugar conversion), which enhances the economic value of OPEFB. A biohydrogen fermentability test of this hydrolysate was carried out using a locally isolated bacterium, Enterobacter sp. KBH6958. The biohydrogen yield after 72h of fermentation was 1.68mol H2 per mol sugar. Butyrate, ethanol, and acetate were the major metabolites.