To illustrate the distribution of chromium （Ⅲ） in the formation of GTF and further reveal the mechanism of chromium rich， this study applied advanced materials science analysis method and biological electron microscope technology. The morphological changes of Saccharomyces cerevisiae were monitored by scanning electron microscope and transmission electron microscope. The distribution in Saccharomyces cerevisiae was revealed by dispersive energy spectrometer-transmission electron microscope （EDS-TEM） and time-of-flight two ion mass spectrometer （TOF-SIMS）. The content of Cr3+ in various parts of Saccharomyces cerevisiae was detected by EDTA elution and enzymolysis. The result shows that with the increase of Cr3+ mass concentration in the fermentation medium， the shape of Saccharomyces cerevisiae changed from regular ellipse to irregular. And when the mass concentration of Cr3+ was high up to 500 μg/mL， the surface precipitation occurs， and the inner cavity appears cavitation. The total chromium and organic chromium content were highest in Saccharomyces cerevisiae， reaching 1 803.87 μg/g·DCW and 1 133.91 μg/g·DCW respectively， when the mass concentration of Cr3+ was 500 μg/mL. 14.43% of the total chromium adsorbed was concentrated in the cell wall， and the other 85.57% was enriched in the protoplasts. In addition， 73.29% of the adsorbed chromium existed as organic chromium in the protoplasts. GTF is mainly formed in the protoplast and only a small amount of inorganic chromium was adsorbed by cell wall of Saccharomyces cerevisiae. And the mass ratio between protoplasts and cell walls is about 5 ∶ 1， we can draw a conclusion that the distribution of GTF in the cell wall or protoplast is approximately homogeneous in Saccharomyces cerevisiae. The results of EDS-TEM and TOF-SIMS further confirms this conclusion.