In this work, during subcritical CO2 pretreatment, the effects of CO2 pressure, reaction time and temperature on compositional changes and subsequent enzymatic hydrolysis were determined. After the pretreatment of 5 MPa CO2, 80 min and 160 °C, the maximal xylose yield was 9.8 g (including 8.4 g oligomer) based on 100 g native material. When the pretreated eucalyptus were enzymatically hydrolyzed for 60 h, 42.4 g glucose based on 100 g native material could be obtained, representing 92.2% of glucose in eucalyptus. Under the most favourable pretreatment and enzymatic hydrolysis conditions, a maximum total sugar recovery of 50.6 g (including 8.5 g xylose and 42.1 g glucose) based on 100 g native material was obtained, which corresponds to 84.1% yield on the basis of total sugar in eucalyptus biomass. Characterization data were obtained for the pretreated eucalyptus using XRD and SEM analyses.