In this paper, a new method was developed for indirect determination of iodine with methyl isobutyl ketone (MIBK) extraction by atomic fluorescence spectrometer (AFS). Based upon the complex, which can be extacted perfectly by MIBK, formed between I- and Hg2+ in 0.1 mol·L-1 of nitric acid, indirectly measuring the iodine in organic phase becomes possible. The effects of extraction conditions and other influence factors were investigated. Under the optimized experimental conditions, the results showed that the linearity between its absorbance and the concentration of iodine was 0～12 μg·L-1, the detection limit is 0.14 μg·L-1, and the relative standard deviation is 3.3%. The method has been applied to the determination of iodine in eggs, and the recoveries are in the range of 101.8%～110.4%.