In this paper, a preparation method of raltitrexed-related impurities was reported, and the effect of related impurities on blood system was studied.The results showed that the method has mild conditions and high yield.The prepared impurities A, B and C are stable quality and can be used to prepare reference materials for the anal. of raltitrexed-related impurities.The effects of impurities A, B and C on blood cells were also studied.The results showed that impurities A, B and C could significantly reduce the number of leukocytes and granulocytes, and their toxicity to the blood system was significantly higher than that of raltitrexed.Therefore, the preparation method of high purity impurities A, B and C can lay a solid foundation for quality research of raltitrexed.