Freeze-drying, continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens. In this study, Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain. The difference between cells preserved with different preservation methods and their initial strains were compared with physiological and biochemical methods and through antibiotics resistance analysis. The composition of protectants for freeze-drying V. anguillarum was optimized. We found that the optimal composition of protectants was 8% of trehalose, 12% of skim milk, 8.0% of lactose, 2.0% of sodium citrate, 12.0% of serum and 8.0% of mannitol. The indexes of lysine decarboxylase and urease changed after continuous passage. The urease reaction changed after freeze-drying and freeze-thawing, but the reaction can be restored to the initial after freeze-drying. Based on the antibiotics resistance analyses, the sensitivity of V. anguillarum to different drugs including rifampicin, erythrocin, furazolidone, ceftazidime, lomefloxacin, gentamycin, azithromycin, doxycycline, ampicillin, co-trimoxazole and cefoperazone changed after different treatments, and some of these changes can be restored to the original through activation culture. In sum, compared with cryopreservation and continuous passage, the freeze-drying is more sustainable for the long-term preservation of V. anguillarum, which showed a better effect in maintaining the original characteristics of pathogen.