Although the cellular endocytosis or uptake research using confocal laser scanning microscopy (CLSM) and Transwell inserts was frequently reported in experimental cell research and pharmaceutical research, there is little report on cellular transport process based on the same techniques. One of main reasons is that fluorescence of most fluorescent reagents could be clearly and definitely seen under CLSM only when they are internalized into and concentrated within cells. Here, Madin-Darby Canine Kidney (MDCK) cells and Coumarin 6 labeled nanoparticles (C6-NPs) was used as models, a new system was developed to image C6-NPs transport across Transwell filter grown cell monolayer by adding free cells into the basolateral medium and making the Transwell insert semi-permeable membrane visible under CLSM. The transport process could be clearly imaged in real-time and in situ, based on the visualization of Transwell membrane indicating the relative position of cells and nanoparticles, and the free cells in the basolateral medium serving as collector and indicator for the transported nanoparticles. The method was also applied in cell migration study. We believe that the novel approach developed here will be certainly useful not only in exploration of nanoparticle cellular transport mechanism, but also in other cell biological sciences based on CLSM and Transwell.