Glucose tolerance factor (GTF) can effectively regulate sugar metabolism, improve the affinity of insulin to receptor, and improve fat metabolism. Up to now, its chromic value in GTF is unclear, which limited its application in reducing sugar health food. In this paper, extraction of GTF with ammonia and ethanol method from high chromium yeast was optimized. And then glucose tolerance factor (GTF) with high purity was obtained by two stages of gel chromatography. HPLC-ICP-MS was used to analyze chromium valence of GTF. The high chromium yeast was treated with 0.1 mol/L of ammonium for 4 h and then 30% of ethanol for 20 min. The total chromium in this extraction was 133 μg. Compared with ammonia extraction alone, the ratio of chromium to protein was increased by 6 times in this way. After extraction, glucose tolerance factor was purified by tow chromatography consecutively. The purified GTF was analyzed by HPLC-ICP-MS. Its chromatography showed that there was only one peak of chromium in this sample. It appeared in range from 31 s to 90 s, which was in agreement with standard of trivalent chromium. Therefore, it could be concluded that chromium in GTF exist in the state of trivalent. This will provide basis for research and development of high chromium yeast for dietary supplement.