To screen protective agents suitable for freeze-dried fish vaccines, low-field nuclear magnetic resonance (LF-NMR) was applied to analyze the water distribution and mobility in different protective agents after freeze drying. According to the relaxation time of hydrogen protons, resident water can be divided into three states: bound water, immobilized water and free water. Results showed that secondary drying temperature had a great impact on the water distribution in mannitol and sucrose, and with the increase of temperature from 4℃ to 37℃, most free water desorbed and some shifted to bound water. When placed in environment with relative humidity of 43% and temperature of 25℃, most protective agents absorbed water and immobilized it to bound water, except that some bound water in mannitol shifted to free water which desorbed to the environment. Water distribution and mobility in different freeze-drying protective agents were not the same, which indicated their different ability in water binding. This research would be helpful for screening of protective agents of freeze-dried fish vaccines and optimization of freeze-drying process, to form a more stable vaccine product.