Abstract A simple method for the preparation of iodine standard solution was proposed. Iodine disproportionation was completed in strong alkaline environment quickly, inverse-disproportionation of mixed solution occurred in strong acid environment when potassium iodide as a co-solvent and the iodine standard solution was then obtained after standing 36 h. Sodium thiosulfate calibration and vitamin C determination were performed to explore the accuracy of iodine solution concentration. The concentration stability of iodine standard solution was discussed also. The rusults showed that the method have many advantages, such as simple, easy to operate, high accuracy, long storage time, calibration error is less than 0.1% and analysis deviation of samples is less than 0.2%.