The flue-cured tobacco variety K326 was used as the material, a one-year experiment was conducted based on a long-term field trial with 5 years of C and 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 the accumulation of soil microbial residue carbon under the two cropping systems were compared. Results showed that under C, compared to C-B0, C-B1, C-B2 and C-B3 significantly increased soil amino sugar and residue carbon contents (P<0.05) 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 different treatments. Under R, compared to R-B0, soil amino sugar, microbial residue carbon, and the contribution of microbial residue carbon to organic carbon all increased first and then decreased with increasing biochar application rate, with the maximum values under R-B1. Furthermore, under B0 and B1, the values of amino sugar, microbial residue carbon, and the contribution of microbial residue carbon to organic carbon were significantly higher under R than under C (P<0.05). Conversely, under B2 and B3, the contents of amino sugar and microbial residue carbon were significantly higher under C than under R (P<0.05). Mantel test and RDA analysis indicated that soil organic carbon was the primary factor influencing amino sugar and microbial residue carbon contents under C. In conclusion, R combined with biochar application is an effective approach to regulating the stability of soil-tobacco-microbial ecosystem, which can effectively mitigate continuous cropping obstacles in tobacco cultivation and achieving sustainable tobacco production..