Glucansucrases are capable of synthesis of distinct oligosaccharides using different acceptor molecules and in this study lactose derivative hetero-oligosaccharides were shown to be produced by an active glucansucrase in its acceptor reaction with sucrose and whey as the donor-acceptor molecules, respectively. Following the monitoring of the reaction products by TLC analysis, the effects of reaction parameters in terms of enzyme concentration, sucrose/whey ratio and reaction temperature on final oligosaccharide yields (OY) were optimized by Response Surface Methodology (RSM). Results indicated that increment of enzyme concentration and reaction temperature decreased the OY in a particular reaction time whereas the OY increased with the increment of sucrose/whey ratio in the acceptor reactions of glucansucrase E81 with whey as the acceptor molecule. A low level of glucansucrase E81 concentration was sufficient to obtain the highest OY in the tested conditions. These findings demonstrated the importance of the optimisation of the reaction parameters for the acceptor reactions of the glucansucrases.