The electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA was described using a single-walled carbon nanotube (SWCNT)/poly(vinylferrocenium) (PVF+) modified pencil graphite electrode (PGE). The electrochemical oxidation signals of guanine were monitored before and after the interaction between MC and DNA by using differential pulse voltammetry. The effects of DNA and MC concentration and MC interaction time were examined based on the electrode response. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of SWCNT/PVF+ modified and PVF+ modified PGEs. The detection limit corresponded to 625 ng/mL for MC using calf thymus double-stranded DNA immobilized SWCNT/PVF+ modified PGE.