The effects of chemical pretreatments (dilute H2SO4, dilute NaOH, and NH4OH) and biological pretreatments (Coriolus versicolor and Daedalea quercina) on the enzymatic hydrolysis of Eucalyptus were investigated. The results showed that Eucalyptus obtained from different regions possess similar chemical compositions and that the optimum particle sizes for reducing sugar production were 60- to 80-mesh. Contrary to the negative influences of a dilute H2SO4 pretreatment, an alkali pretreatment showed positive effects on Eucalyptus saccharification. This phenomenon may had been attributed to the efficient removal of lignin and the stronger structural damage during the alkali pretreatment process. In comparison with the chemical pretreatments, a higher reducing sugar yield could be achieved from the biological pretreated Eucalyptus. The highest reducing sugar yield of 97.14 mg/g was obtained from the Guangxi (GX) Eucalyptus that was pretreated with Daedalea quercina.