The invention discloses a high-purity temsirolimus represented by the formula (I), the HPLC purity of the temsirolimus can be above 99.5%. The invention also provides a method for preparing high-purity temsirolimus by virtue of a preparative high performance liquid chromatographic separation system. The method comprises, but not limited to the sequence of the steps, the following steps of A) normal-phase high performance liquid chromatography and B) reverse phase high performance liquid chromatographic separation, wherein in the step A), gradient elution is carried out by taking a silicon ball filler as a stationary phase, ethyl acetate of which the concentration is 60-100v/v% and n-hexane or n-heptane of which the concentration is 40-0v/v% as mobile phases; in step B), gradient elution is carried out by taking C4, C6, C8 or C18 fillers as stationary phases and acetonitrile and aqueous solution of which the pH value is 3.5-6.0 as mobile phases; and the temperature of the system is 10-30 DEG C. The process is simple and environmentally friendly and high-purity and high-quality temsirolimus can be obtained by virtue of the process.