Isotope analysis of trace uranium is important in nuclear safeguards and nuclear forensics, which requires the analytical methodologies with high sensitivity, accuracy, and precision. As one of the most powerful techniques in isotopic measurement, thermal ionization mass spectrometry (TIMS) usually suffers from its relatively low sensitivity in ultratrace measurements. To overcome this limitation, we have developed a new filament carburization technique for TIMS, with graphene oxide (GO) as the ionization enhancer. A high and steady ionization efficiency of ∼0.2% for uranium was achieved in single-filament mode, which was 10× the classical double-filament method. With total evaporation (TE) measurements, this method was validated with certified reference materials (CRMs) at the picogram level, and the relative uncertainties for n(235U)/ n(238U) were as low as the ∼1% level. The enhancement mechanism of GO's promoting effect on uranium ionization was attributed to the uniform microstructure facilitating energy transfer and formation of carbides. This approach provides an alternative simple and rapid method for trace uranium isotope analysis with high sensitivity and excellent repeatability. Filament carburization and uranium loading could be accomplished within 10 min. This technique has great advantage in analysis of trace uranium isotope ratios and can be applied in the researches of environmental analysis and nuclear forensics.