The limited information on biological fate impedes the development of more efficient polymeric nanoparticles for oral delivery of bio-macromolecules. In this study, the in vivo fate as well as the trans-epithelia transport of polycaprolactone (PCL) nanoparticles is explored by labeling with aggregation-caused quenching probes, which is capable of identifying intact nanoparticles. Live imaging and confocal laser scan microscopy confirm size-dependent absorption of PCL nanoparticles. In general, reducing particle size favors a faster and more oral absorption. Nanoparticles larger than 200 nm, such as 600 and 2000 nm, cannot be efficiently transported across the intestinal membrane. The absorbed nanoparticles (50 and 200 nm) mainly accumulate in the liver. Lymph may be the main absorption route for PCL nanoparticles, transporting 2.39 ± 1.81% and 0.98 ± 0.58% of administered 50 and 200 nm nanoparticles, respectively. Cellular uptake and transportation of PCL nanoparticles are also size dependent. Both enterocytes and M cells mediated transcytosis are involved in the transport of 50 nm PCL nanoparticles, while the M cell pathway is dominative for other nanoparticles. In conclusion, the study provides a valuable tool for bioimaging of intact polymeric nanoparticles as well as solid evidence supporting size-dependent translocation of the nanoparticles via oral delivery.