A rapid and simple co-precipitation method for iodine separation from natural samples was developed for determination of 129I using accelerator mass spectrometry (AMS). The method includes three steps, i.e. decomposition of organic iodine, iodine separation and target preparation using co-precipitation, and sensitive measurement of 129I/127I ratio using AMS. The recovery of iodine was higher than 90% for most natural water samples in the decomposition step using K2S2O8. Iodine in the digested sample was then co-precipitated as AgI-AgCl-Ag2SO3-Ag2SO4 with a typical recovery of 95-98%. It was observed that addition of 0.2 mg 127I carrier can significantly improve the measurement uncertainty of low-level 129I samples by enhancing iodine ion current. The overall recovery of iodine in the entire procedure was higher than 80%, and a detection limit of 1.0×105 atoms (or 2.2×10-17 g) for 129I was achieved. The developed method was validated by certified reference material (IAEA-418) and five types of natural water samples in comparison with results using the conventional solvent extraction method. Analytical results showed good agreement with the reference values for all samples, confirming the reliability of the developed method. The simple operation, with no need for organic solvent and small amount of iodine carrier addition make the developed method suitable for rapid and reliable determination of low level 129I in natural water samples.