Development of an efficient intraocular drug delivery nanosystem remains the most difficult challenge to attain a prolonged therapeutic effect at the site of drug action. The purpose of this work was to develop a biodegradable, long-term sustained release, and biocompatible nanoparticulate system to treat various intraocular diseases. To attain the objectives, poly(ortho ester) (POE), a hydrophobic, surface erodible, and nontoxic polymer, was selected for fabrication of nanoparticles for the first time using a double emulsion solvent evaporation method. The influence of POE molecular weight on particle size, polydispersity index, zeta potential, drug content, in vitro release, degradation, in vitro cytotoxicity, and cell uptake studies was investigated. Drug-loaded nanoparticles had a spherical shape with an average particle diameter from 241 to 298 nm and zeta potential values from -8 to -11 mV. Encapsulation efficiency ranged between 21 and 63%, depending on the type of the water-soluble molecule used. Approximately 20-30% of the loaded drug was released over a period of 14 weeks. The drug release and degradation profiles of nanoparticles followed perfect zero-order kinetics confirming the POE-surface erosion mechanism. In vitro cytotoxicity and cell uptake studies revealed the cyto-compatible nature and nonendocytic behavior of POE nanoparticles. Collectively, POE nanoparticles are a very promising vehicle for sustained delivery of therapeutics to the back of the eye.