This work presents a new chemosensor Labetalol (LBT) hydrochloride 5-[1-hydroxy-2-(1-methyl-3-phenylpropylamino)ethyl]salicylamide hydrochloride (LBT) was applied for spectrophotometric determination of UO22+ in aqueous solution. The chemosensor LBT react with UO₂²⁺ forming a new complex UO₂²⁺/LBT at wavelength 314 nm. The absorbance of the linear calibration curve increases by increasing UO₂²⁺ concentration at linear range of (1.0 – 16.1) × 10−6 mol L-1 with correlation coefficient (R2 = 0.998). The experimental conditions which affecting the absorbance development were studied and optimized. The complexes stoichiometry was measured at the absorbance spectra 314 nm and the results were found to be 1:1 (metal: ligand) ratio. The present chemosensor LBT was effectively validated with respect to linearity, precision, accuracy, detection and quantification limits. For more accurate analysis, the apparent molar absorptivity, Beer's law, Ringbom and Sandell's sensitivity have been studied. Results of analysis were statistically compared with those obtained by ICP-OES as a reference method show that, the Student's t- and F-values at 95% confidence level are less than the theoretical values, which confirmed that there is no significant difference between the chemosensor LBT and the reference method in the same studied conditions. The chemosensor LBT provide a simple, sensitive and inexpensive spectrophotometric determination without any complicated equipment. Also it was successfully applied to directly the determination of UO22+ in different aqueous samples with the satisfactory results.