[Background] Leptospirillum ferrodiazotrophum can produce a large amount of secondary minerals during the culture. [Objective] This study optimized the culture conditions of L. ferrodiazotrophum for secondary mineral production, aiming to provide materials for the subsequent application of biogenic secondary minerals. [Methods] Single factor tests were carried out to investigate the effects of pH, temperature, rotation speed, and liquid loading on the production of secondary minerals by L. ferrodiazotrophum, on the basis of which the response surface method was employed to optimize the culture conditions. Subsequently, the produced secondary minerals were characterized and tested. [Results] The single factor tests showed that the secondary mineral yield reached the highest under the conditions of pH 3.00, 35.0 °C, and rotation speed of 150 r/min, and the effect of liquid loading on the secondary mineral production was insignificant. The optimal conditions for producing secondary minerals were pH 2.82, 31.7 °C, and rotation speed of 135 r/min. After the optimization, the yield increased from the highest value of 57.714 mg/(L·h) in the single factor tests to 65.436 mg/(L·h). X-ray diffraction results showed that the produced mineral had poor crystallinity and was an amorphous hydroxyl high-iron mineral. The mineral had a short needle-like structure and a large specific surface area. [Conclusion] This study optimized the biosynthesis conditions of secondary minerals through single factor tests and response surface design, laying a foundation for applying secondary minerals in the future.