Abstract: Long-term continuous cropping of adlay ( Coix lacryma-jobi L.) and the indiscriminate application of chemical fertilizers have led to soil fertility declines and acidification. To explore the effects of tobacco stalk biochar-based fertilizer on the soil organic carbon (SOC) fractions and microbial community structure and abundance, tobacco stalk biochar-based fertilizer was used in a field experiment with four treatments: no fertilizer, conventional fertilizer, low tobacco stalk biochar-based fertilizer, and high tobacco stalk biochar-based fertilizer. Changes in the activities of four enzymes related to soil carbon cycling and microbial activity were evaluated, and the relationships between the soil pH, SOC fractions, soil enzymes, and soil bacterial abundance were analyzed. The results showed that: 1) The application of tobacco stalk carbon-based fertilizer significantly increased the soil pH and the contents of SOC, dissolved organic carbon (DOC), particulate organic carbon (POC), and microbial biomass carbon (MBC) (P<0.05). The MBC was most affected, increasing by 41.09%−76.04% compared to conventional fertilizer application. 2) The application of tobacco stalk biochar-based fertilizer significantly increased the activities of soil amylase and dehydrogenase (P<0.05). Compared to conventional chemical fertilizers, amylase and dehydrogenase activities increased by 44.28% and 57.54%, respectively, whereas the soil invertase activity was unaffected when tobacco stalk biochar-based fertilizer was applied. 3) The application of tobacco stalk biochar-based fertilizer increased the Chao1 and Shannon indexes, abundance and diversity of the soil bacterial communities. 4) The application of tobacco stalk biochar-based fertilizer affected the composition and structure of the soil bacterial community, increased the relative abundance of Actinomycetes and Bacteroides, and reduced the relative abundance of Proteobacteria and Chloroflexus. It also significantly increased the abundance of Nitrospira, Bryobacter, and other bacterial genera, and significantly reduced the abundance of Aciditerrimonas and Crenothrix. 5) Redundancy anaylsis showed that soil pH, carbon fraction, soil enzymes activities, and soil bacterial community abundance were correlated each other after the application of tobacco stalk biochar-based fertilizer; soil pH, SOC, POC, DOC, MBC were significantly positively correlated with the activities of various soil enzymes (P<0.05), but were significantly negatively correlated with Proteobacteria (P<0.05). In summary, tobacco stalk biochar-based fertilizer increased the soil pH, SOC fractions, soil enzymes activities, and soil bacterial abundance, which improved the soil bacterial community structure and the adlay planting soil and optimized the soil ecology. This study provides a reference for the resource utilization of tobacco stalk and improvements in soil fertility.