This paper focuses on the dilution agent effect onto interactions between methylene blue (MB) and DNA using carbon fiber microelectrode (CFME) based DNA biosensor. In this study, designed CFME based DNA biosensor was successfully carried out considering the effect of probe dilution agent (e.g. 3-mercaptopropionic acid) against proposed hybridization mechanism types. The voltammetric signals of MB were measured at bare CFME, 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 probe dilution agent is a significant factor in determining the type of DNA hybridization mechanisms.