Aptamers, the short single-stranded oligonucleotide or peptide sequences isolated via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) have empowered the development of aptamer-based biosensors for the detection of protein biomarkers. Following the discovery of SELEX, the thrombin-binding aptamers have been extensively studied and frequently used to demonstrate the proof-of-concept of the aptasensors. Abnormality of thrombin, the dominating enzyme in the blood coagulation cascade is considered a tumor biomarker and is known to be associated with thromboembolic disorders, arteriosclerosis, Alzheimer’s disease, neuroinflammatory diseases, etc. Hence, the development of highly sensitive and selective detection methods of thrombin is of paramount importance in clinical research and diagnosis for the understanding, treatment, and prevention of a multitude of diseases. Among the various aptasensing techniques employed for thrombin detection, electrochemical aptasensors have received tremendous attention due to the simple instrumentation, rapid response, inherent miniaturization, robustness, independence of sample turbidity, and cost-effectiveness. In this chapter, the emphasis has been placed on the recent advances in electrochemical aptasensors for thrombin detection. In addition to the discussion on some commonly conjugated nanomaterials in aptasensing for signal amplification and sensitivity improvement, this chapter also imparts valuable information regarding SELEX, the common immobilization techniques of aptamer, and the clinical importance of thrombin.