Efficient transfection of molecular beacons has to be performed in the microscale in order to fully utilize the potential of molecular beacons and microfluidics for studying the real-time gene-expression dynamics in living cells. Nevertheless, there has been relatively little study on transfection of molecular beacons in microfluidic channels.In this work, the differences between transfection in conventional cell culture systems and in microfluidic cell culture systems were investigated systematically with a combination of computational and experimental methods. Comparison between a no-flow microchannel and a 96-well plate revealed that the scale-dependence of reaction-diffusion kinetics contributes to the reduced transfection efficiency in the no-flow microchannel. Study on transfection in the microfluidic system under flow conditions suggested that the fluid flow enhances mass transfer, while the fluid shear stress can reduce the transfection efficiency.The results of this study will provide useful guidelines in optimizing molecular beacon transfection efficiency in microfluidic systems for studying gene-expression dynamics in living cells.