In the present study, we describe the isolation and detailed characterization of pepsin-soluble atelocollagen from Rhizostoma pulmo species jellyfish and application towards thrombin apta-sensing. Various analysis methods including infra-red spectroscopy, SDS-PAGE electrophoresis, and amino acid analysis have been applied for the characterization of jellyfish collagen and compared with both rat tail collagen and BSA. When comparing the two collagen types derived from jellyfish and rat tail, jellyfish collagen was observed to contain a relatively high amount of glutamic acid (61 residues/1000 residues) and alanine (63 residues/1000 residues) but low amounts of proline (113 residues/1000 residues). On the other hand, pepsin-soluble jellyfish collagen contained a small quantity of tyrosine indicating the purity of atelo-collagen. Electrochemical impedance spectroscopy is the main analyzing technique of the developed apta-sensor. The proposed apta-sensor has a detection limit of 6.25 nM thrombin. Clinical application were performed with analysis of the thrombin levels in blood and CSF samples obtained from patients with Multiple Sclerosis, Myastenia Gravis, Epilepsy, Parkinson, Polyneuropathy and healthy donors using both the apta-sensor and commercial ELISA kit. The results revealed the proposed system to be a promising candidate for clinical analysis of thrombin. (C) 2016 Elsevier B.V. All rights reserved.