Herein, a unique and versatile immobilization free elctrochernical nucleic acid biosensor architecture is proposed for the first time based on the catalyzed release of a methylene blue (MB)-tagged mononucleotide by exonuclease III (Exo III) and the successive enrichment Onto a dodecanethiol monolayer, which can be attributed to the hydrophobic force between the alkyl chain of the dodecanethiol monolayer and the hydrophobic part of the MB -tagged mononudeotide. The fabricated biosensor demonstrates considerable advantages including assay simplicity, rapidness, and high sensitivity owing to its immobilization-free and homogenolis operation for the biorecognition and amplification process. A low detection limit of approximately 1 pM toward the target DNA could be achieved with an excellent selectivity. The proposed immobilization-free electrochemical biosensing strategy was also extended for the 'assay,of Exo I and III activity. Furthermore, it might be easily extended for the detection of a wide spectrum of targets and thus provide a pthrnising avenue for the development of immobilization-free and sensitive electrochemical biosensors.