Thermal ionization mass spectrometry (TIMS) is extensively used for uranium assay and isotopic measurements in various aqueous samples, but it is a multistep process involving sample pretreatment, separation, preconcentration of uranium from the matrix, loading on a rhenium filament for TIMS analysis, and so forth. The present work reports the synthesis of bis(2-ethylhexyl)phosphoric acid (DEHPA)-functionalized pore-filled membranes that offer a single-step, fast, and efficient extraction of uranium from natural water samples. The equilibrium sorption capacity of the DEHPA pore-filled membrane for uranium, in pH = 6, was found to be 31 mg/g of the membrane, and the sorption equilibrium was achieved within 70 min. The membrane also served as a loading substrate for uranium isotopic analysis by TIMS and significantly enhanced the production of UO + ions, compared to the U + ions. With the developed methodology, it was possible to measure the 234 U/ 238 U isotope ratio in natural water samples with precision as better as 0.15%, without demanding any state-of-the-art instruments and, at the same time, without compromising the accuracy and precision in the measurement of the minor abundant isotope, that is, 234 U. Also, the analysis time was reduced to >80% compared to the conventional TIMS technique involving ion-exchange/extraction chromatographic columns, and a detection limit of about 3 ppb was achieved.