The rice-crayfish (RC) integrated farming system has been recognized for improving spatial efficiency, diversifying food chains, and enhancing biodiversity. However, the long-term impacts of RC co-culture on soil organic carbon stock (SOCS), total nitrogen stock (TNS) and soil quality index (SQI), across different soil depths remain poorly understood. Thus, we conducted an 8-year field study in the Jianghan Plain, comparing the SOCS and SQI in the topsoil (0-20 cm) and subsoil (20-40 cm) of rice monoculture (RM) and the RC system. Our findings indicate that the RC system increased SQI by 38.6% and SOCS by 27.5% in the topsoil, while it increased TNS by 15.9% and SQI by 17.5% in the subsoil. The RC system enhanced soil pH, SOC, dissolved organic carbon (DOC), and available phosphorus (AP) across the 0-40 cm profile, and raised the concentrations of ammonium nitrogen (NH 4 +-N) and dissolved organic nitrogen (DON) in the topsoil. Conversely, RC decreased DON and bulk density (BD) in the subsoil. Additionally, RC reduced all three soil enzyme activities (sucrose hydrolase, nitrate reductase, and phosphatase enzyme) in both topsoil and subsoil, suggesting that changes in the soil environment, such as flooding, induced by rice-crayfish integrated farming may negatively impact soil health. Furthermore, we observed a positive correlation between soil quality and soil nutrients, indicating that the diversified residue input from the RC system may be crucial for improving soil quality by enhancing soil nutrients, metabolic potential, and carbon sequestration. Our study underscores the potential of the RC system to enhance soil quality and carbon sequestration, offering valuable insights for sustainable agricultural practices and long-term soil management.