Two-stage solid-state fermentation strategy was exploited and systematically optimized to enhance Bacillus subtilis growth and sporulation for increasing effective cell number in B. subtilis microbial ecological agents. The first stage focused on improving cell growth followed by the second stage aiming to enhance both cell growth and sporulation. The optimal fermentation condition was that temperature changed from 37 °C to 47 °C at a fermentation time of 48 h and Mn²⁺ content in medium was 4.9 mg MnSO₄/g dry medium. Solid medium properties were improved by the optimal two-stage fermentation. HPLC results demonstrated that glucose utilization was facilitated and low-field nuclear magnetic resonance (LF-NMR) results showed that more active sites in medium for microbial cells were generated during the optimal two-stage fermentation. Moreover, microbial growth and sporulation were enhanced simultaneously during the second stage of fermentation through delaying microbial decline phase and increasing sporulation rate. As a result, effective cell number of B. subtilis reached 1.79 × 10¹⁰/g dry medium after fermentation for 72 h, which was 29.7% and 8.48% higher than that of conventional fermentation for 72 h and 48 h, respectively. Therefore, the optimal two-stage fermentation could increase the effective cell number of B. subtilis microbial ecological agents efficiently.