This study investigates the effects of inoculum ratio, substrate particle size, and aeration rate on humic acid (HA) biosynthesis during aerobic composting of rice straw. The optimal conditions for HA biosynthesis were found to be an inoculum ratio of 20%, a substrate particle size of 0.83 mm, and an aeration rate of 0.3 L·kg⁻¹·DM·min⁻¹, resulting in a maximum HA yield of 356.9 g kg⁻¹. The study also analyzed the microbial community changes and metabolic functions during composting. The dominant phyla were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with Rhizobium, Phenylobacterium, Pseudoxanthomonas, and Paenibacillus positively related to HA content. The metabolic function profiles indicated that genes involved in carbohydrate and amino acid metabolism were involved in lignocellulose biodegradation and HA biosynthesis. The results suggest that optimizing these operational parameters can enhance the bioconversion efficiency of agricultural residues into biofertilizers. The study highlights the importance of microbial diversity and metabolic functions in the composting process for effective HA biosynthesis and the utilization of agricultural residues.This study investigates the effects of inoculum ratio, substrate particle size, and aeration rate on humic acid (HA) biosynthesis during aerobic composting of rice straw. The optimal conditions for HA biosynthesis were found to be an inoculum ratio of 20%, a substrate particle size of 0.83 mm, and an aeration rate of 0.3 L·kg⁻¹·DM·min⁻¹, resulting in a maximum HA yield of 356.9 g kg⁻¹. The study also analyzed the microbial community changes and metabolic functions during composting. The dominant phyla were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with Rhizobium, Phenylobacterium, Pseudoxanthomonas, and Paenibacillus positively related to HA content. The metabolic function profiles indicated that genes involved in carbohydrate and amino acid metabolism were involved in lignocellulose biodegradation and HA biosynthesis. The results suggest that optimizing these operational parameters can enhance the bioconversion efficiency of agricultural residues into biofertilizers. The study highlights the importance of microbial diversity and metabolic functions in the composting process for effective HA biosynthesis and the utilization of agricultural residues.