Thrombin overexpression in serum serves as a critical biomarker and is implicated in several diseases associated with significant morbidity and mortality. Existing techniques for thrombin detection are time-consuming and require sophisticated equipment and extensive sample preparation procedures, which further delay the detection and increase the cost of the procedure. Early and accessible diagnosis at the point of care, especially in limited-resource countries, represents the first step of clinical interventions. To overcome these limitations, we have proposed an innovative, sustainable paper-based electrochemical detection platform for thrombin. In this work, a sustainable paper-based aptasensor was rationally designed, characterized, evaluated against conventional gold standard plastic-based substrates, and applied to human serum, yielding a detection limit of ~ 60 pM. The present method provides an efficient and user-friendly way for the detection of thrombin and potentially leading to better management and treatment outcomes for patients.