Enzymatic biodiesel production was investigated in solvent-free and isooctane systems using Burkholderia cenocepacia lipase (BCL) immobilized on macroporous resin NKA as a biocatalyst. A total of 98% biodiesel yield was obtained under the optimized conditions of methanol/oil molar ratio 4:1 with the addition of methanol in three steps at intervals of 2 h, enzyme dosage 2.5 wt % (based on the oil weight), moisture concentration 7 wt % (based on oil weight), reaction temperature 40 degrees C, reaction duration 8 h, stirring rate 300 rpm, and isooctane amount 50 wt % (based on the oil weight). Compared to tert-butanol and solvent-free systems, the highest biodiesel yield was achieved in the isooctane system. BCL-NKA showed high operational stability with no obvious loss in lipase activity for transesterification in the isooctane system even after 50 cycles (400 h) of repeated usage. It has been revealed that BCL-NKA-catalyzed transesterification in the isooctane system is a promising alternative for biodiesel production.