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A novel bio-fertilizer, produced from prickly ash seeds (PAS), <i>Bacillus subtilis</i> and biochar, was evaluated for its disease-preventing potential on tobacco black shank caused by <i>Phytophthora nicotianae</i>. The results showed that biochar promoted the growth of Tpb55 in PAS and increased the pH of the organic fertilizer. The final concentration of <i>B. subtilis</i> could reach 1.7 × 10¹⁰ cfu g⁻¹ in the biological organic fertilizer (PBB) under the optimal medium under conditions of solid-state fermentation. PBB exhibited a strong fumigation effect on <i>P. nicotianae</i>, including inhibiting mycelium growth, reducing the disease severity and decreasing the pathogen population in rhizospheric soil. PBB treatment also could significantly increase the pH of acidified soil and improve soil nutrition content such as available K, alkali hydrolysable N and organic carbon. High-throughput pyrosequencing of 16S and 18S rRNA genes revealed that 4% PBB addition in soil had significant effects on the diversity and richness of fungi but not on that of bacteria. The microbial community structure was also shifted after PBB treatment. Some potentially beneficial microbes such as <i>Bacillus</i>, <i>Mucor, Cunninghamella, Chitinophaga</i> and <i>Phenylobacterium</i> were enriched, while potential pathogen <i>Fusarium</i> was significantly decreased. In conclusion, the agricultural waste PAS combined with biochar can replace soybean as a source for the production of biocontrol <i>B. subtilis</i> Tpb55, and the novel bio-fertilizer could effectively control tobacco black shank by pathogen inhibition, soil nutrient improvement and shifting the rhizomicrobial community.