Characteristics of cellulose isolated from oil palm mesocarp fiber (OPMF) using multi-step pretreatment methods were determined in this study. It was postulated that the succession of pretreatment steps for cellulose extraction from lignocellulosic biomass may affect the cellulose properties. Pretreatment steps involved in cellulose extraction were superheated steam (SHS) treatment, enzymatic hydrolysis and alkaline treatment. OPMF was subjected to SHS treatment to open up the fibres wall structure followed by enzymatic hydrolysis using xylanase for effectiveness enhancement the alkaline treatment afterwards. In order to obtain bleached cellulose, peracetic acid, a totally chlorine free (TCF) chemical was used. Cellulose obtained from these multi-step pretreatment methods had purity of 87.5 %, with crystallinity of 69.2 % and thermally stable up to 360°C.