This study investigated the response of microbial residues in flue-cured tobacco rhizosphere soil under continuous cropping and crop rotation to biochar application, providing new insights into mitigating continuous cropping obstacles and improving soil quality using corn straw biochar. Using the flue-cured tobacco variety K326 as the material, a one-year experiment was conducted based on a long-term field trial with 5 years of continuous cropping (C) and rotation (R). Four biochar application rates were set: B0 (0 kg/hm2), B1 (300 kg/hm2), B2 (750 kg/hm2), and B3 (1500 kg/hm2). The effects of the four biochar treatments on soil microbial residue carbon accumulation under the two cropping systems were compared. Results showed that under continuous cropping, compared to C-B0, soil amino sugar and residue carbon contents significantly increased (P<0.05) in all treatments, following the order C-B0 < C-B1 < C-B2 < C-B3. Specifically, C-B1, C-B2, and C-B3 treatments increased total amino sugar and microbial residue carbon by 13.26%, 24.22%, 36.20%, and 10.53%, 20.23%, 32.89%, respectively. However, there was no significant difference in the contribution of microbial residue carbon to organic carbon among treatments. Under the crop rotation system, compared to R-B0, the soil amino sugar, microbial residue carbon, and the contribution of microbial residue carbon to organic carbon showed a trend of increasing and then decreasing with increasing biochar application rates, with the maximum value observed in treatment R-B1. Furthermore, under B0 and B1 treatments, the values of amino sugar, microbial residue carbon, and the contribution of microbial residue carbon to organic carbon were significantly higher in crop rotation than in continuous cropping (P<0.05). Conversely, under B2 and B3 treatments, the contents of amino sugar and microbial residue carbon were significantly higher in continuous cropping than in crop rotation (P<0.05). Mantel test and RDA analysis indicated that soil organic carbon was the primary factor influencing amino sugar and microbial residue carbon content in continuous cropping soil. In conclusion, crop rotation combined with biochar application is an effective approach to regulating the stability of the soil-tobacco-microbial ecosystem in production practice, effectively mitigating continuous cropping obstacles in tobacco cultivation and achieving sustainable tobacco production.