Abstract: The aim of this study was to evaluate the potential application of layered double hydroxide(LDH)nanosheets for ocular drug delivery. Using LDH nanosheets as carriers, carboxymethyl cellulose(CMC)as a stabilizer and pirenoxine sodium(PRN)as the model drug, CMC-PRN-LDH nanosheets were prepared. PRN-LDHs nanoparticles were synthesized via co-precipitation method. X-ray diffraction, atomic force microscopy, transmission electron microscopy and laser particle sizer were employed to characterize the physicochemical properties of LDH nanosheets, CMC-LDH nanosheets and PRN-LDH nanocomposites. Stability, accumulative release in vitro and precorneal retention in vivo of both CMC-PRN-LDH nanosheets and PRN-LDHs nanoparticles were evaluated. It was found that CMC-PRN-LDH nanosheets were electrostatically stabilized by CMC absorbed on the surface of LDH nanosheets, but PRN-LDHs nanoparticles aggregated in phosphate buffered saline. 12-hr accumulative release percentage of PRN from CMC-PRN-LDH nanosheets and PRN-LDHs nanoparticles were 70. 44% and 44. 21% in vitro, respectively. Compared with the commercial PRN eye drops, there existed 4. 18-fold increase in AUC_{0-6 h} and 1. 79-fold in mean retention time of CMC-PRN-LDH nanosheets. Negligible levels of PRN-LDHs nanoparticles might be attributed to inter-groups difference. Draize test showed that CMC-PRN-LDH nanosheets were non-irritant to the rabbit eyes after single and repeated dosing. It suggest that this novel LDH nanosheet could be a promising carrier for ocular drug delivery with prolonged residence time.