Larch dieback disease, caused by Neofusicoccum laricinum, severely affects forest health and productivity. To effectively curb the occurrence of this disease, a powder formulation of Bacillus amyloliquefaciens JL54 was developed through spray drying. The research commenced with the optimization of fermentation medium and culture conditions through statistical design, aiming to maximize both bacterial viability and antagonistic activity. The optimal medium included 12 g/L yeast extract, 11.8 g/L yeast powder, and 7.2 g/L magnesium sulfate. Optimal culture conditions included 30% loading volume, 1% inoculum, 37 °C incubation temperature, 31.8 h shaking time, and initial pH 6.4. Under these conditions, the viable count of strain JL54 reached 4.45 × 10⁹ cfu/mL, a 296.67-fold increase compared with the unoptimized system. To evaluate its practical applicability, field trials were conducted, showing a 54% control efficiency against larch dieback disease, significantly suppressing disease progression. Subsequently, the spray drying process was optimized with a 1:10 protective agent-to-water ratio, 100 °C inlet temperature, and 630 mL/h feed flow rate, achieving a 78.41% powder recovery rate. Collectively, this study demonstrates the potential of B. amyloliquefaciens JL54 as an effective biocontrol agent for managing larch dieback and supports its application in spray-dried formulations for forest disease control.