The enzymatic transesterification of ethyl ferulate (EF) with cod liver fish oil was investigated with Novozym 435 as catalyst under solvent-free conditions. The purpose of the study is to evaluate the synthesis system for production of feruloyl fish oil in industry. The modified HPLC method was firstly set up to characterise the reaction products together with HPLC-ESI-MS. The adding of glycerol to the system on the profile of feruloyl acylglycerol species was investigated in terms of transesterification performance. The bioconversion rate of EF can be significantly increased with increased formation of feruloyl fish oil products as well when appropriate amount of glycerol was present in the reaction. Therefore, the addition of equivalent molar amount of glycerol to EF was decided for the practical optimization of the system. The mutual effects of temperature (40 to 70 oC), reaction time (1 to 5 days), enzyme load (2 to 20 %) and substrate amount ratio of fish oil/EF (1 to 5) were thus studied with assistance of response surface methodology (RSM) for the purpose of maximizing the formation towards feruloyl fish oil. The models were well fitted and verified. The optimized conditions were found to be: temperature, 70 °C; enzyme load, 4.3 %; substrate ratio, 4.7; and reaction time, 5 days. Under these conditions, the maximum conversion of EF could reach 92.4 %, and formation of feruloyl fish oil could reach 80.4 %. But the formation of by product was minimized to 11.4 % only.