Abstract: Nutrient leaching can occur after large-scale flood irrigation of biogas slurry. Soil degradation has also been found after the long-term excessive application of chemical fertilizers. An optimal nutrient strategy is often required to design the biogas slurry application machinery. In this research, a systematic investigation was implemented to explore the effects of various biogas slurry application modes on raw soil fertility and soybean seed quality. A fertilization regime was combined to select the organic fertilizer as the basal application and biogas slurry as the topdressing. An isonitrogenous experiment comprised six treatments of biogas slurry application, including root watering application (JS), foliar spraying application (PS), a combined half-watering and half-spraying application (JP), shallow injection at a depth of 5 cm (5ZS), middle injection at a depth of 10 cm (10ZS), and deep injection at a depth of 15 cm (15ZS). A field experiment was conducted to systematically evaluate the differential impacts of these application modalities on soil physicochemical properties, heavy metal accumulation profiles, soil microbial community diversity, soybean agronomic growth traits, and final seed nutritional quality. The experimental results demonstrated that different application modes significantly remodelled the root zone microenvironment for the nutrient uptake and partitioning patterns in soybeans, which ultimately determined the seasonal yield and quality. Specifically, there was the short-term yield-increasing surface application. The JS treatment achieved the highest available potassium content in the topsoil (293.05 mg/kg). Consequently, there were the superior soybean yields (2146.03 kg/hm²) and seed crude fat content (24.82%), and the crude protein content maintained at a high level (34.38%). While the lower soil organic matter (only 4.45 g/kg) resulted in the lower contents of seed microelement, such as iron (Fe) and manganese (Mn), compared with the injection. The JP treatment effectively enhanced the topsoil available phosphorus content (17.57 mg/kg) and the surface soil organic matter. The high seasonal yields were promoted the synergistic accumulation of zinc (Zn) and copper (Cu) in the seeds. Both JS and JP were induced the negative environmental effects, including surface soluble salt accumulation, the structural degradation of soil macro-aggregates, and an apparent depletion of the total phosphorus nutrient pool. Conversely, the middle and deep injection was enhanced the soil carbon sequestration, fertility and crop quality. The 10ZS treatment was used to stimulate the effective nitrogen supply potential of the soil. The highest levels of topsoil alkaline hydrolyzable nitrogen and organic matter were achieved in the entire experiment (53.68 mg/kg and 10.17 g/kg, respectively). The 10ZS significantly increased the accumulation of trace elements (Fe, Mn, Cu, and Zn) in the soybean seeds, due to the localized zone with the high-concentration nutrient supply. Furthermore, the best performance of 15ZS treatment was obtained to enhance the diversity of the soil microbial community. Differentiated strategies were given in practical production, according to the specific soil conditions. The JS treatment was preferred for the high seasonal yields, providing that efficient application equipment was developed to mitigate surface salinity. Alternatively, the 10ZS or 15ZS with the basal organic fertilizer was strongly recommended to improve the soil structure, organic matter, and microbial diversity. Precision-positioning injection machinery can be developed with adjustable depth and in-situ salinity monitoring to circumvent nutrient leaching risks.