Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.