Abstract: Objective: Glycerolysis of rapeseed oil was catalyzed with lipozyme TL IM lipase to produce diacylglycerol (DG). Methodology: To optimize DG yield, the effect of five-level four factors and their reciprocal interactions on product yield through Response Surface Methodology (RSM) was evaluated. Thirty individual experiments were performed to investigate reaction temperature, reaction time, enzyme loading and substrate mass ratio. Results: Well-fitting model was established through multiple regression with backward elimination for DG yield (R2 = 0.8388). The optimal conditions established include 60°C reaction temperature, 8 h reaction time, 16% enzyme loading and 0.07 substrate mass ratio (glycerol to rapeseed oil), resulting to 82.4% DG yield. Conclusion: The experimental data showed good validation with the predicted value. Transesterification of rapeseed oil and glycerol was monitored by using Fourier transform infrared (FT-IR) spectroscopy.