a This work was aimed to synthesize and characterize poly(2-hydroxyethyl methacrylate) [poly (HEMA)]-based molecularly imprinted polymer nanoparticles (MIP NPs) containing timolol maleate (TM) via precipitation polymerization. The molecular structures of the MIP and non-imprinted polymer (NIP) NPs were compared by means of Fourier transform infrared spectroscopy. The morphological observations by using scanning electron microscopy and transmission electron microscopy confirmed the formation of MIP NPs as small as 128 nm in average diameter with appropriate synthesis conditions. Thermal behaviors of the samples were also studied by the use of thermogravimetric analysis and differential scanning calorimetry. By considering a series of key factors such as monomer : template ratio, cross-linker type, pH, and temperature, the sample with promising characteristics was found to be that of HEMA : TM ratio of 10:1, 40 mmol of ethylene glycol dimethacrylate as cross-linker, and polymerization temperature of 60°C in acetonitrile as porogenic solvent. Furthermore, the ultraviolet-visible (UV-vis) spectrophotometry results proved a controlled release of TM from the MIP NP samples compared with NIP ones at extended periods. Moreover, the cytotoxicity of the MIP and NIP NPs samples was evaluated on mesenchymal stem cells, and the obtained observations showed that they had no adverse side effect on the living cells; especially the surface of the MIP NPs sample depicted highly cellˈs biocompatibility. Finally, the outcomes from designed different experiments conducted us that the HEMA-based MIP NPs have great potential as an ocular nanocarrier for TM delivery.