Objective To explore and establish the pretreatment and determination of iodine in breast milk. Methods The breast milk samples were digested by a mixture of concentrated nitric acid and hydrogen peroxide (HNO₃- H₂O₂ ) in the sealed vessel, then the iodine in digestion solution was subjected to the analysis by arsenic cerium catalytic spectrophotometry. Results The standard curve range of iodine concentration was 0-300 μg/L, the correlation coefficient of standard curve was more than 0.9999,the minimum detectable concentration was 8 μg/L (Sampling volume of 5.00 ml), the recovery of standard addition was between 86.5% and 102.6%, and the relative standard deviation of high, medium and low concentration was less than 4%. The average iodine of 900 breast milk was 169.2 μg/L. Conclusion The method of microwave digestion-arsenic cerium catalytic spectrophotometry determination of iodine in breast milk could digest the sample thoroughly and this method had good reproducibility, high sensitivity, and good accuracy, suitable for the determination of iodine in breast milk.