Shewanella haliotis has potential biotechnological applications in shrimp culturing and oil pollution abatement. To determine the optimal fermentation medium and culture conditions of Shewanella haliotis, single factor experiments and orthogonal design were conducted. Further confirmation was carried out in 70 L bioreactor under the optimal medium and culture conditions. The results showed that the optimal medium was 6% sucrose, 2.5% peptone and 0.7% CaCO₃ at initial pH 6.8, and optimal culture conditions in shaking flask were 30℃, 130 r/min, 100 mL/500 mL loading volume, 6% inoculum size and 24 h fermentation cycle. The average of final biomass in 70 L bioreactor reached 1.14×10¹¹ cfu/mL. The fermentation optimization provides experiment data for further industrial production.