Oil Palm Fruit Mesocarp (OPFM) is a solid by-product containing cellulose, potentially serving as a raw material for biofuel. The cellulose content of this solid by-product can be extracted through delignification. Therefore, this study aimed to investigate the application of ozone for OPFM delignification to break down lignin bonds in the material. During the analysis, ozonolysis delignification was influenced by particle size, oxygen flow rate, and reaction time. Ozone flowrate analyzed using the Iodometric method. Cellulose, hemicellulose, and lignin content of raw material and treated samples were analyzed using the α-cellulose, γ-cellulose, and the Kappa method. The results showed that by using a particle size of 100 mesh, and a flow rate of 2 Lmin-1 for 15 min, ozone degraded 42.03% lignin, 15.89% hemicellulose, and concentrated 62.85% cellulose. SEM and FTIR results showed the removal of hemicellulose and lignin from OPFM with ozonolysis delignification. Furthermore, XRD analysis showed the crystallinity degree of the high cellulose yield.