Two-dimensional (2D) layered semiconductors with excellent electrical, optical, physical, and chemical properties have been extensively investigated over the past decade. Beyond graphene and transition metal dichalcogenides (TMDCs), 2D perovskites have also garnered significant research interest owing to their unique optical and electrical properties. Among 2D perovskites, 2D lead-free double perovskites with inherent stability and remarkable electrical performance are promising for electronic applications. Most immunosensors and traditional bioassays require specialized cold chain systems to maintain biorecognition elements. This study presents the preparation of a 2D perovskite-based biosensor and the integration of a biopolymer to fabricate biopolymerpreserved biosensors with enhanced thermal stability for the sensitive detection of cardiac troponin I (cTnI). This sensing device showed remarkable sensing performance for the detection of cardiac troponin I, with a detection limit of 10 pg/mL. Furthermore, this energy-efficient approach maintained the biosensing capability of the 2D perovskite-based biosensor at elevated temperatures.