This paper reports a systematic and detailed investigation on the interactions between methylene blue (MB) and DNA immobilized onto carbon fiber microelectrode (CFME). In this study, designed CFME based DNA biosensor was successfully carried out considering three different hybridization mechanisms to obtain more specific and selective results. The consistency of the results was tested with the various probe configurations (unlabeled and amino-labeled). The voltammetric signals of MB were measured single stranded DNA (ssDNA)-modified CFME and double stranded DNA (dsDNA)modified CFME by means of square wave voltammetry (SWV). The electrochemical parameters for MB on binding to DNA onto single CFME in the solution and at the electrode surface were described. This study shows that before or after hybridization, MB accumulation is related with probe configuration. It provides very useful information for developing carbon fiber based sequence-specific electrochemical DNA sensors.