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With the large-scale cultivation of transgenic Bacillus thuringiensis (Bt) crops, the Bt toxin released from Bt crops is continuously introduced into the soil. Its environmental fate represents a key indicator for assessing the ecological safety of transgenic crops. However, the persistence of Bt toxin in soil is influenced by both biotic and abiotic processes, and their respective contributions under natural conditions remain unclear. This study measured water-dissolved Bt toxin concentrations in paddy soil (PS) and red soil (RS) to compare the influence of biotic and abiotic factors on the dynamic retention of exogenous Bt toxin under different sterilization methods: no sterilization, heat sterilization (HT), and irradiation sterilization (IS). The water-dissolved Bt toxin exhibited a dynamic decrease–increase–decrease trend across all three treatments in both soil types during the 30 day experimental period. Bt toxin displayed rapid adsorption during the initial 2 h stage in RS, but subsequently showed a high desorption, whereas PS probably achieved more stable bonding through soil organic matter (SOM). Different sterilization methods significantly influenced the results by altering abiotic factors: Compared to CK, HT affected soil physicochemical properties and enhanced adsorption resilience, whereas IS caused minimal impact on the soil physicochemical properties, thereby providing a more accurate reflection of abiotic processes. And microbial, as biotic facters, also influence the reduction process of Bt toxin by participating in the adsorption–desorption–degradation equilibrium process. Therefore, we infer that over time, the concentration of water-soluble Bt proteins in the soil will tend toward zero. Additionally, the initial Bt toxin concentration influenced dynamic balance by adjusting adsorption site saturability, with more pronounced desorption reversibility at 500 ng/g concentrations. Overall, this study systematically reveals the effects of soil properties, microorganisms, and sterilization methods on Bt toxin persistence. The findings underscore the importance of selecting and justifying sterilization methods in related environmental behavior studies, while providing essential guidance for the scientific assessment of environmental risks posed by transgenic crops.