A rapid method based on extractive electrospray ionization mass spectrometry (EESI-MS) has been developed for quantitative detection of trace radioactive molecular iodine-129 ( 129 I 2 ) in negative ion detection mode in ambient air. For actual air samples, gaseous 129 I 2 was completely converted into iodine-129 ions ( 129 I À ) by an excess of Na 2 SO 3 solution. By adding excess amounts of 127 I 2 into the solution, the 129 I À ions formed triiodide ion complexes (i.e. 129+127*2 I 3 À ) which were subjected to tandem mass spectrometry experiments to exclude false positives. Quantification of iodine-129 was achieved by quantitatively measuring the characteristic fragment (i.e., 129 I À ) of the triiodide ion complexes. The calibration curve showed a good linearity within an relatively wide concentration range of 0.01-1000 ppb (R 2 ¼ 0.991), a limit of detection (LOD) of 4.5 ppt and a relative standard deviation (RSD) of 4.0-13.1% (n ¼ 5). The spiking recovery of this method was found to be 82.6-110.5%. The method had also been successfully applied to detecting the trace amount of gaseous 129 I 2 released in a simulated nuclear leakage accident, showing a satisfactory result for the tested gaseous samples. The experimental data demonstrated that EESI-MS was a useful tool for quantitative measurement of radioactive iodine in cases such as nuclear leakage, nuclear explosions and related scenarios.