The purpose of the present study was to prepare and characterize twice-daily sustained-release matrix tablets of Timolol maleate (TM) using different concentrations of natural, synthetic and semi-synthetic polymers. The effect of nature of the diluents and method of preparation were also studied. Formulations were evaluated for the release of TM over a period of 12 hours using United States Pharmacopoeia (USP) type-II dissolution apparatus. Along with physical properties, the dynamics of water uptake and erosion degree of tablets were also studied. The in-vitro drug release study revealed that the most successful formulation of the study F28 (drug to polymer ratio 1:2) which includes both HPMC K100 and EC (1:1), extended the drug release up to 12 hours, exhibited satisfactory drug release in the initial hours, and the total release pattern was close to the theoretical release profile with similarity factor (f2) above 50. The drug release from optimized formulation (F28) followed first-order kinetics via non-Fickian anomalous) diffusion. FTIR studies revealed that there was no interaction between the drug and excipients. In conclusion, the results indicated that the prepared sustained-release tablets of TM could perform therapeutically better than conventional tablets with improved efficacy and better patient compliance.