The purpose of this study was to prepare ginkgolide B (GB) lyophilized powder for injection with excellent appearance and stable quality through a formulation screening and by optimizing the freeze-drying process. Cremophor EL as a solubilizer, PEG 400 as a latent solvent, and mannitol as an excipient were mixed to increase the solubility of GB in water to more than 18 times (about from 2.5×10-4mol/L (0.106mg/mL) to 1.914mg/mL). Formulation screening was conducted by orthogonal design where the content of GB in the solution before lyophilization (using external standard method of HPLC) and reconstitution time after lyophilization were the two evaluation indexes. The optimized formulations were GB in an amount of 2mg/mL, Cremophor EL in an amount of 16% (v/v), PEG 400 in an amount of 9% (v/v), mannitol in an amount of 8% (w/v), and the solution pH of 6.5. Through four single-factor experiments (GB adding order, preparation temperature of GB solution, adding amount, and adsorption time of activated carbon), the preparation process of GB solution was confirmed. The glass transition temperature of maximally GB freeze-concentrated solution was -17.6°C through the electric resistance method. GB lyophilized powder began to collapse at -14.0°C, and the fully collapsed temperature was -13.0°C, which were determined by freeze-drying microscope. When the collapse temperature was determined, the primary drying temperature was obtained. Thereby, the freeze-drying curve of GB lyophilized powder was initially identified. The freeze-drying process was optimized by orthogonal design, the qualified product appearance and residual moisture content were the two evaluation indexes. The optimized process parameters and process were (1) shelf temperature, decreased from room temperature to -45.0°C, at 0.5°C/min in 2h; (2) shelf temperature increased from -45.0 to -25.0°C, at 0.1°C/min, maintained for 3h, and the chamber pressure was held at 10Pa; (3) shelf temperature was increased from -25.0 to -15.0°C at 0.1°C/min, maintained for 4h, and the chamber pressure was held at 10Pa; and (4) shelf temperature was increased from -15.0 to 20.0°C at 1.0°C/min, maintained for 4h, and the chamber pressure was raised up to 80Pa. In these lyophilization process conditions, the products complied with relevant provisions of the lyophilized powders for injection. Meanwhile, the reproducibility was satisfactory. Post-freezing annealing had no significantly beneficial effects on shortening the freeze-drying cycle and improving the quality of GB lyophilized powder.