Excessive use of inorganic fertilizers disrupts soil nutrient balance and leads to soil degradation and a decrease in biodiversity. In contrast, bio-fertilizers enhance soil structure and fertility and promote plant growth and sustainable agriculture development. Therefore, this study focused on a rotation system of winter wheat and summer maize and aimed to explore the effects of applying chemical fertilizer (NPK) and bio-fertilizer (BF) in the winter wheat season on the sustainable soil development of current wheat and subsequent maize. Before sowing winter wheat four fertilization treatments were, respectively CK (100% NPK at 750 kg ha−1), A (60% NPK at 450 + 20% BF at 150 kg ha−1), B (60% NPK at 450 + 40% BF at 300 kg ha−1), and C (60% NPK at 450 + 60% BF at 450 kg ha−1), conducted. The results showed that treatment A (60% NPK + 20% BF) replacing the NPK at 300 kg ha−1 with BF at 150 kg ha−1 significantly soil nutrient contents, enzyme activity, and microbial metabolic activity. The study also found a positive correlation between soil parameters (total nitrogen, alkaline nitrogen, available phosphorus, organic matter, urease, and alkaline phosphatase in the winter wheat and maize cropping season). Furthermore, the soil microbial composition showed significant enrichment of Proteobacteria, Acidobacteria, Actinobacteria, and Firmicutes, and variations among treatments. Moreover, the application of biofertilizer enhanced the diversity of soil fungi species, particularly during the winter wheat season. This study highlights the importance of integrating biofertilizers with NPK fertilizer for agricultural system conversion and promoting agricultural production and sustainability.