In-situ non-invasive observation of concentration polarization (CP) and fouling layer is of vital importance to understanding membrane filtration process and membrane permeability loss. This study developed a novel real-time technique, laser induced fluorescence (LW) technique, to visualize CP development and fouling layer formation, and to measure solute concentration field on the membrane surface directly during cross-flow nanofiltration, which helped to quantitatively evaluate the effect of various operation conditions on membrane permeability. The solute concentration distribution on the membrane surface was successfully determined from the LW images, and the fouling layer thickness was measured by series of processed images after binary subtraction. LIF results demonstrated that the fouling development experienced approximate two stages, i.e. rapid growth phase and consolidation phase, and visualized the transition from initial CP to the later fouling layer formation. Afterwards, the formed fouling layer tend to be steady with nearly unchanging concentration and thickness. Quantitative analysis proved that the operational hydraulic condition has a significant influence on the formation and stabilization of fouling layer. The developed method and obtained results were very useful to in-depth recognize the nanofiltration process and to understand the membrane fouling mechanism.