Abstract: We obtained high quantity small RNA from the mesocarp of oil palm (Elaeis guineensis) and screened fatty acid biosynthesis related miRNAs. The extracted method of CTAB was improved and then employed to isolate small RNA from the mesocarp of oil palm nuts at five development stages (G1-G5). Twelve miRNAs were screened from our previous database of oil palm small RNA using bioinformatics analysis. Relative expression of each miRNA was determined by stem-loop real-time quantitative PCR(qPCR) and further predicted the targets of these miRNAs. The results indicated that the OD₂₆₀/OD₂₈₀ values of the small RNA were between 1.7 and 2.0, and the concentrations of G1 to G5 were 289 ng/μL,364 ng/μL, 476 ng/μL, 213 ng/μL, and 390 ng/μL, respectively. Relative expression detected by qPCR showed that the twelve miRNAs exhibited significantly different expression during the five development stages, especially high expression level in the fourth stage (G4) and fifth stage (G5). Among them, miR395 and miR156 were highest in the G4 phase and miR395 and miR528 were highest in the G5 phase.Target prediction suggested that some targets of these differently expressed miRNAs were involved in the fatty acid metabolism pathway, such as phosphatidate, phosphatase and phospholipase D. In this study, high quantity small RNA was isolated by the improved CTAB method. qPCR further determined that the twelve candidate miRNAs were differently expressed during the five development stages, of which five miRNAs may be involved in the regulatory pathway of fatty acid metabolism.