||DNA-based bio-analysis is important in the fields of molecular diagnostics, environmental and food safety monitoring, pathogen identification, forensic sciences, etc. Electrochemical DNA-based bio-analysis has the advantages of high sensitivity, compatibility for miniaturization and easy operation. Most of existing electrochemical approaches require a probe immobilization step, which increases preparation time, lowers hybridization efficiency, requires washing steps, hampers multiplexing and therefore limits the application of the technology in point-of-care scenarios. This thesis describes a novel “immobilization-free” approach for electrochemical DNA-based bio-analysis in which no probe immobilization step is required and the hybridization or other interaction between the detection probe and the target occurs in a homogeneous solution phase. The approach takes advantage of the neutral backbone of a ferrocene-labeled peptide nucleic acid (Fc-PNA) probe, utilizing the electrostatic repulsion between the negatively-charged DNA backbone and a negatively-charged ITO electrode to transduce the hybridization between the target DNA and the Fc-PNA into a reduction of the electrochemical signal of Fc, therefore realizing an immobilization-free, signal-off electrochemical DNA detection. Based on the immobilization-free electrochemical DNA detection, an electrochemical melting curve analysis technique is developed, as well as a multiplex immobilization-free electrochemical DNA detection and quantification method. In order to produce more reliable results, the immobilization-free electrochemical DNA detection is transformed to the “signal-on” operating mode by introducing an additional DNA probe and solution-phase competitive hybridization into the system. Furthermore, the application of the immobilization-free electrochemical DNA detection strategy is expanded to the detection of DNA-modifying enzymes (exemplified with DNA polymerase) and aptamer-binding bio-molecules (exemplified with adenosine). Finally, the detection sensitivity of the immobilization-free electrochemical DNA-based bio-analytical platform is improved by using an electrode coated with a positively-charged conducing polymer as well as an interdigitated array electrode. The development of an immobilization-free detection platform is a great leap towards point-of-care applications of electrochemical DNA-based bio-analysis technologies.