The integrity of poly(ethylene glycol)-co-poly(e-caprolactone) (PEG-PCL) micelles transcellular transported across madin-darby canine kidney (MDCK) epithelial cells was investigated. Fluorescein isothiocyanate isomer I (FITC) was conjugated to PEG-PCL and the product PEG-PCL-FITC was identified by fluorescence spectra. Two micelles were prepared using the thin-film hydration method: 3,3′-dioctadecyloxacarbo-cyanine Perchlorate (DiO) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine Perchlorate (DiI) co-loaded PEG-PCL micelles (DiO-DiI-M), DiI loaded and PEG-PCL-FITC contained micelles (FITC-DiI-M). The size of the micelles was characterized by dynamic light scattering analysis using a Malvern Zetasizer Nano ZS and it turned out that the particle sizes of both micelles were about 30 nm with identical polydispersity index (PDI). The stability of the micelles in phosphate buffer saline (PBS) was monitored using fluorescence spectra and both micelles were stable within 4 h in PBS. The integrity of PEG-PCL micelles in the transcellular process across MDCK epithelial cell monolayer at 1 and 4 h was investigated using laser confocal scanning microscope and Förster resonance energy transfer (FRET) technology. The Person's coefficient and FRET efficiency of both Transwell layer and Receive layer were recorded. The results show that the FRET efficiency and Person's coefficient of the Receive layer was consistent with that of Transwell layer for both the micelles at 1 h, but decreased at 4 h and FITC-DiI-M decreased more significantly than DiO-DiI-M. The results indicated that the micelles could transport across the MDCK monolayer intactly at 1 h but some of them were disassembled during the 4 h transportation process.