Nanofiltration, as a technology with low energy consumption, no secondary pollution, and continuous and compact operation, is widely used in the fields of seawater desalination, resource recovery and reuse, and drinking water purification, etc. However, “trade-off” effect and membrane fouling are the main problems limiting the application of nanofiltration membranes. Herein, glycerol was introduced into the aqueous reactive solution in interfacial polymerization (IP) for regulating chemical composition and membrane structure simultaneously. As glycerol raised the viscosity of the reactive solution which slowing down the diffusion rate of amine monomer, crumpled structure generated by the diffusion-driven instability. At the same time, the addition of glycerol varied the oxygen content in the skin layer of the membrane. At 60% glycerol content, the as-prepared membrane achieved a pure water permeance of 21.14 L·m−2·h−1·bar−1 with sodium sulfate rejection of 95.6%. However, the oxygen content in the chemical composition of the membrane showed a minimum value with the increase of glycerol concentration indicating a competitive reaction between the amine monomer and glycerol. Through the anti-fouling analysis of membranes with different chemical compositions and structures, it was found that the increase in roughness of nanofiltration membranes with similar chemical composition resulted in an increase of irreversible pollution. The nanofiltration membrane with similar membrane structure and higher oxygen content had stronger anti-fouling ability to humic acid. It is concluded that the rational design of the crumpled membrane with high oxygen-chemical composition is expected to obtain a high flux nanofiltration membrane with anti-fouling performance.