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. High-throughput sequencing revealed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant phyla, with their relative abundances varying over time. Key bacterial genera, such as *Rhizobium*, *Phenylobacterium*, *Pseudoaxanthomonas*, and *Paenibacillus*, were positively correlated with HA content. Metabolic function profiles indicated that carbohydrate and amino acid metabolism genes were involved in lignocellulose biodegradation and HA biosynthesis. These findings provide insights into the microbial mechanisms underlying HA biosynthesis and offer potential strategies for improving the bioconversion efficiency of agricultural residues into biofertilizers.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. High-throughput sequencing revealed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant phyla, with their relative abundances varying over time. Key bacterial genera, such as *Rhizobium*, *Phenylobacterium*, *Pseudoaxanthomonas*, and *Paenibacillus*, were positively correlated with HA content. Metabolic function profiles indicated that carbohydrate and amino acid metabolism genes were involved in lignocellulose biodegradation and HA biosynthesis. These findings provide insights into the microbial mechanisms underlying HA biosynthesis and offer potential strategies for improving the bioconversion efficiency of agricultural residues into biofertilizers.