Molecular dynamics simulation has been widely used in the membrane separation techniques. In order to further reveal the mechanism of nanofiltration separation of monosaccharides, molecular dynamics method was used to simulate the diffusion process of eight monosaccharides in the aromatic polyamide nanofiltration membrane. Furthermore, the interaction energy between membrane material and monosaccharides was also investigated. Simulation results showed that the interaction force between monosaccharides and membrane material was in the order of sorbose > fructose > glucose > mannose > galactose, and ribose > xylose > arabinose. The diffusion coefficient of eight monosaccharides inside the membrane was in the order of sorbose > galactose > glucose > mannose > fructose > ribose > xylose > arabinose. Within the same experimental conditions, the rejection rate sequences of eight monosaccharides were in the order of: fructose > mannose > glucose > galactose > sorbose > arabinose > xylose > ribose. This finding indicated that solute radius has a clear effect on solute retention. The diffusion ability of solute molecules and the interaction with membrane material also influence the separation of monosaccharides through nanofiltration. A strong interaction force outside the membrane enables many monosaccharide molecules to be adsorbed in the surface of membrane and then driven into the membrane pores which can contribute to the reduction of rejection. Rejection rates were negatively related to the diffusion coefficient of monosaccharides inside the membrane. Monosaccharides can easily pass through the membrane pores under high diffusion ability, thus leading to low rejection rates of monosaccharides.