Fe (Ⅲ) hydrolysis is an important process during geochemical circulation of iron, and controls the migration and redistribution of iron in water to some extent. The existing form of Fe (Ⅲ) in supernatant and the difference of Fe isotopic composition between the supernatant and the precipitate caused by Fe (Ⅲ) hydrolysis at 20℃ and 46℃ were studied in this paper. The experimental time lasted 95 days and 130 days at 20℃. At the end of this experiment, the existing form of Fe (Ⅲ) was colloid, Fe isotopic composition in different experimental periods were the same, and the difference of Fe isotopic composition between the supernatant and the precipitate (Δ⁵⁶Fe_{Fe(Ⅲ)sup-Fe(Ⅲ)pre}) was 1.15‰. The experimental time lasted 95 days at 46℃. At the end of this experiment, the existing form of Fe(Ⅲ) was aqueous, and the difference of Fe isotopic composition between the supernatant and the precipitate (Δ⁵⁶Fe_{Fe(Ⅲ)sup-Fe(Ⅲ)pre}) was 1.37‰. The instantaneous equilibrium fractionation factor (α) of Fe(Ⅲ) hydrolysis could be calculated by Rayleigh fractionation formula, and the α of 20℃ and 46℃ were 0.999 121 and 0.999 260 respectively.