A novel isolation of oil palm empty fruit bunch lignocellulose nanofibers (LCNFs) was successfully applied using multimechanical stages assisted with different vibration milling times. Morphology, physical, chemical, and thermal properties of LCNFs were studied. All LCNF samples were agglomerated due to hornification process during the drying of its suspension. The external surface of the nanofibers was irregular, uneven, folding, and unsmooth with approximate nanofibers diameter of above 100 nm. The optimal size of oil palm empty bunch LCNFs was in the range of 53.72–446.80 nm. Bio-oil was suspended in all LCNF samples after analysis with GC–MS in which the most dominant extractives were aromatic and oxygenated compounds. The increase in milling time could lead to the decrease in silica and oxygen elemental compositions. IR analysis revealed that there were no pronounced changes of transmittance peaks during vibration milling processes. The increase in milling time induced the decrease in atomic crystal size of LCNFs but the increase in crystallinity index from 7 to 17 milling times. A pretreatment of 7 vibration milling times to LCNFs brought about the lowest thermal stability.