A novel method in the radioimmunoassay (RIA) technique was developed to attain the optimum conditions for the estimation of thyroid hormones, triiodothyronine (T3) and thyroxin (T4), by applying kinetic and thermodynamic studies. Polystyrene beads were coated by specific antibodies (Ab) of both T3 and T4 hormones at the same time, to create immobilized solid phase available for binding with antigens (Ag) and 125I-labeled antigens (Ag*) by forming immunocomplexes according to RIA measurements. Kinetic investigations were conducted by applying the initial rate technique, with the orders determined and the rate equations deduced. Thermodynamic studies included the determination of ΔG 0, ΔH 0, and ΔS 0. They revealed that the reactions proceed spontaneously and are endothermic with entropy gain, which is characteristic of inner-sphere complexation. The positive ΔH 0 value matched with the determined activation energy (E a). Forty samples of different thyroid status were assayed for T3 and T4 using the present system and with commercially available kits. The statistical analysis revealed good correlations between them. This work may offer an easier and more efficient method in diagnostics and treatment of thyroid dysfunctions.