In this paper, the method of extraction and transient transformation of oil palm leaf protoplasts was established. Protoplasts were isolated from leaves of oil palm by 30 g/L cellulase and 8 g/L macerozyme for 3.5 h, and collected by centrifugation at 2000 rpm/min for 5 min at 4°C. Moreover, the dual plasmid transient co-expression system was optimized using green fluorescent protein (GFP) and red fluorescent protein (RFP). The best transformation efficiency was the obtained when the plasmids were mixed with oil palm protoplasts with the mass ratio of 8: 1, heat shocked at 45 °C for 20 min after placed on ice for 30 min, and incubated at room temperature in the dark for 30 min with equal amount of PEG/Mg2+ (200 g/L PEG 4000 and 100 g/L MgCl2). Finally, the protoplast isolation and transient expression system provides a new method for the functional verification of the genes form oil palm.