This study explores the immobilization of porcine trypsin (PT) on superparamagnetic iron oxide nanoparticles (SPIONs) via adsorption, with a focus on both immobilization conditions and a comprehensive energetic evaluationan aspect often neglected in the literature. Isothermal titration calorimetry (ITC) revealed that interactions at pH 8.0 are energetically favorable, with a ΔH° of −43.0 kJ mol^–1, suggesting robust noncovalent interactions. PM6 calculations confirmed the stability of the adsorption. The resulting nanobiocatalyst showed over 75% activity recovery at pH 8.0 and retained around 40% activity after four reuse cycles, demonstrating its efficiency and robustness. The detailed energetic analysis provides critical insights for optimizing immobilization processes, impacting cost and technical feasibility, and advancing the understanding of enzyme-support interactions for scalable applications in biocatalysis.