In this study, chemical pulping techniques were applied to create a set of biomass substrates with intact lignocellulosic fibers and controlled morphological and chemical properties to allow the investigation of the individual effects of xylan and surface lignin content on enzymatic hydrolysis. A high resolution X-ray photoelectron spectroscopy technique was established for quantifying surface lignin content on lignocellulosic biomass substrates. The results from this study show that, apart from its hindrance effect, xylan can facilitate cellulose fibril swelling and thus create more accessible surface area, which improves enzyme and substrate interactions. Surface lignin has a direct impact on enzyme adsorption kinetics and hydrolysis rate. Advanced understanding of xylan and surface lignin effects provides critical information for developing more effective biomass conversion process. Published by Elsevier Ltd.