Organic farming is considered a sustainable agricultural practice that can enhance soil health and reduce environmental pollution. However, the long-term effect of organic farming on soil quality, microbial structure and functions, as well as the underly mechanism still remains incomplete. Here, we investigated the effect of 5, 15, and 20 years of organic farming on soil chemical and biological properties at 0–20 cm (topsoil) and 20–40 cm (subsoil) depths in comparison with conventional farming in a subtropical region. The Soil quality index (SQI) was calculated using a total dataset of soil nutrients (i e., SOC, TN, TP, etc.), enzyme activities (C- and N-acquiring enzymes), and microbial diversity (Fungal chao1, bacterial chao1). The results showed that organic farming significantly increased soil quality by 53–103 % in topsoil and by 25–142 % in subsoil. Organic farming increased soil total and available nutrients, soil water content, C- and N-acquiring enzyme activities, and microbial diversity. Compared to conventional farming, the enzyme activities for C- and N- acquisition in organic farming increased by 3 times and 2 times, respectively, and stabilized over time. Microbial community analysis indicated a rich and complex network under long-term organic management, with a notable increase in the number of nodes and edges (doubled after 20-year), signifying a more resilient ecosystem. In conclusion, long-term organic farming significantly enhances soil quality and microbial diversity, with the most substantial improvements observed in longer duration, thereby supporting the sustainability of agricultural practices in subtropical regions. The study underscores the importance of long-term organic farming in improving soil quality and provides scientific evidence for advocating sustainable agricultural practices in subtropical regions. © 2024 Elsevier B.V.