A hydrophobic deep eutectic solvent (HDES)-based optode was designed for the preconcentration and determination of the UO22+ ion in aqueous media using spectroscopic techniques [energy-dispersive X-ray fluorescence (EDXRF) and solid-state absorption]. The optode was developed by incorporation of HDES (tri-n-octyl phosphine oxide and decanoic acid in an equimolar ratio), tri-(2-ethylhexyl) phosphate, and 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol into a cellulose triacetate matrix. Characterization studies were carried out using different techniques to understand the roles of HDES as a plasticizer, UO22+ extractant, and Br-PADAP immobilizer. Uptake studies revealed that the optimal pH was 3 and sorption followed the type II adsorption isotherm. Uranium in the U-sorbed optode can be directly analyzed over a large concentration range of 0.021 × 10-3-2.1 × 10-3 Mol L-1 using EDXRF. The optode film exhibited a linear dynamic range of 0.84 × 10-6-84 × 10-6 Mol L-1 for uranium, with a lowest limit of detection of 0.084 × 10-6 Mol L-1 by colorimetric analysis. This optode-based method was employed for seawater analysis for its UO22+ concentration without any matrix separation, and the concentration was found to be 1.30 ± 0.06 × 10-8 Mol L-1. The optode exhibited better selectivity for UO22+ in the presence of various cations including Sr2+ and Cs+ in an aqueous medium. Compared to other prevailing optical sensors, this optode performed better in terms of key factors like pH, equilibration time, reusability, and detection limit.