Abstract: Biological nitrogen fixation is a major nitrogen source in alfalfa fields, and the nitrogen supply and soil fertility can be largely affected by the composition and quantity of the nitrogen-fixing bacterial community. In this study, a field experiment was conducted to explore the soil nitrogen-fixing bacterial community structure and abundance characteristics in loessal soil with different alfalfa growing ages (2, 9 and 18 years planted in 2019, 2012, and 2003, respectively), using farmland (maize field) as the control. The fluorogenic quantitative real-time PCR technique was adopted in the experiment, using the high-throughput sequencing platform Illumina MiSeq to target the nifH gene. We analyzed the ecological status of abundant and rare nitrogen-fixing microorganisms through co-occurrence networks and identified the dominant factors affecting the community structure of nitrogen-fixing microorganisms by soil coupling the physical and chemical properties. The results showed that long-term planting of alfalfa increased the organic carbon, total nitrogen, and soluble carbon contents of the soil. The nifH gene abundance ranged from 2.97×10⁶ copies∙g⁻¹ to 5.93×10⁶ copies∙g⁻¹ in dry soil and was significantly higher in alfalfa fields than in farmland. The correlation analysis between the abundance of nifH gene of nitrogen-fixing microorganisms and soil physicochemical factors showed that nifH gene abundance in the soil was positively correlated with bulk density (P=0.009) and soluble carbon content (P=0.005), positively correlated with total nitrogen (P=0.044) and available potassium (P=0.013) contents, and negatively correlated with total phosphorus content (P=0.000) and nitrate content (P=0.023). A total of 176 367 valid sequences were obtained, belonging to five phyla, eight classes, 11 orders, 15 families, and 17 genera. Proteobacteria and Cyanobacteria were the dominant phyla, accounting for 95.9%−98.9% and 0.2%−1.8% of the total sequences of the samples, whereas Skermanella and Azohydromonas were the dominant genera, accounting for 82.2%–87.6% and 1.6%–4.6%, respectively. Compared with farmland, continuous alfalfa planting significantly increased the relative abundance of Skermanella, but its’ relative abundance decreased with increasing alfalfa planting years. Long-term cultivation of alfalfa propagated microbial taxa, including Azotobacter, Burkholderia, Frankia, Mesorhizobium, Geobacter, and Bradyrhizobium; whereas Clostridium, Rhodopseudomonas, and Trichormus were sterilized. Redundancy analysis (RDA) showed niche differentiation for the nitrogen-fixing bacterial community in response to environmental factors, but total phosphorus, organic carbon, and nitrate-nitrogen in the soil were the dominant environmental factors for the nitrogen-fixing bacterial community structure. Analysis of the molecular ecological network showed that there were 520 nodes and 4170 edges in the network of nitrogen-fixing microorganisms in maize fields and alfalfa soil, among which 24 nodes belonged to the abundant group, 93 nodes belonged to the rare group, and 403 nodes belonged to the transitional group. There was one internal connection of abundant taxa, 2187 internal connections of transitional taxa, and 358 internal connections of rare taxa. Nitrogen-fixing bacteria have a cooperative relationship in their ecological network, with a relatively stable community structure and strong adaptability to environmental changes. This study provides basic data and a theoretical basis for the diversity of nitrogen-fixing microorganisms in loess soil and the determination of a suitable planting period for alfalfa.