This investigation intended to optimize the culture medium for Priestia paraflexa ONG1, a fish probiotic, by combining conventional methods with factorial experimental designs. One-variable-at-a-time (OVAT) experiments demonstrated that lactose and tryptone were the preferred carbon and nitrogen sources, respectively, whereas optimal growth occurred at pH 7.6. Plackett-Burman design (PBD) was employed to screen seven variables (lactose, tryptone, yeast extract, NaCl, KH₂PO₄, MgSO₄, and MnSO₄) for their influence on probiotic cell density. Statistical analysis revealed yeast extract, MgSO₄, and MnSO₄ as significant factors (p < 0.05), which were subsequently optimized using response surface methodology (RSM). Optimal concentrations for the three factors were identified using a Box-Behnken design (BBD). A predictive model for cell density was then generated using a quadratic equation. The model exhibited a strong fit (R² = 98.73%, adjusted R² = 96.45%) and identified yeast extract, MnSO₄, and their quadratic and interaction terms as the most influential factors (p < 0.05). The optimized medium, containing lactose (20 g/L), tryptone (2 g/L), yeast extract (8.82 g/L), NaCl (2.5 g/L), KH₂PO₄ (1.25 g/L), MgSO₄ (0.15 g/L), and MnSO₄ (0.5 g/L), resulted in a maximum cell density of 6.5 × 10⁹ CFU/ml, representing a 28.3-fold increase compared to the control nutrient broth medium. This study demonstrated the successful optimization of growth conditions for P. paraflexa ONG1, providing valuable insights for the development of this promising aquaculture probiotic.