Old oil palm trunk (OPT) is available in large quantity in Southeast Asia and a potential lignocellulosic biomass resource for bioethanol production. Dilute acid (DA) pretreatment was applied to the old oil palm trunk for enzymatic saccharification. The pretreatment conditions were investigated through a fractional factorial experiment design. The pretreated substrates were analyzed for chemical composition, and their enzymatic digestibility was investigated and compared. The results indicated that the DA pretreatment was able to improve enzymatic hydrolysis by removing hemicelluloses from OPT. Mild pretreatment preserved more hemicellulose and cellulose in pretreated OPT, but severe pretreatment was necessary to achieve satisfactory enzymatic hydrolysis of OPT. For example, the DA pretreatment with 3% H2SO4 at 180 °C for 40 min could achieve an 80% enzymatic hydrolysis