The objective of the research was to investigate the effect of biomass loading, acid concentration and pre-treatment time on the yield of sugars obtained after acid pre-treatment and enzymatic hydrolysis of oilseed rape (OSR) straw. The highest concentration of glucose (313.4 ± 7.53 mg g−1 biomass) extracted after hydrolysis was achieved when OSR straw was pre-treated for 90 min; a glucan conversion efficiency of 81%. The highest concentration of sugars extracted immediately after pre-treatment was achieved with a pre-treatment time of 60 min. Pre-treatment energy efficiency in terms of total yield of sugars per MJ of energy consumed was higher when OSR straw was pre-treated for 60 min compared to 90 min even though the conversion of sugar extracted was lower at 60 min.