The invention provides a freeze-dried powder injection of pemetrexed dipotassium and a preparation method thereof. Specifically, the freeze-dried powder injection of pemetrexed dipotassium comprises pemetrexed dipotassium and mannitol and comprises an anti-oxidant optionally. The preparation method comprises the following steps: mixing pemetrexed dipotassium with a KOH solution at first so as to obtain a pemetrexed dipotassium solution; then uniformly mixing aqueous solutions of accessory materials and adding the obtained mixture into the pemetrexed dipotassium solution; and carrying out degerming and freeze-drying so as to obtain the freeze-dried powder injection of pemetrexed dipotassium. The prepared freeze-dried powder injection of pemetrexed dipotassium has the advantages of good stability, low moisture content, good redissolvability, low impurity content and controllable quality. The preparation method is simple and applicable to industrial production.