Gemifloxacin (GMF) is a broad-spectrum quinolone antibacterial agent used in the treatment of acute bacterial exacerbation of chronic bronchitis and mild-to-moderate pneumonia. The investigation of the GMF - double-stranded DNA (dsDNA) interaction using an electrochemical DNA-biosensor, showed for the first time clear evidence of interaction with dsDNA. The interaction between GMF and dsDNA onto pencil graphite electrode (PGE) was assessed at pH 4.80 acetate buffer using differential pulse voltammetry (DPV). The interaction mechanism was disclosed with the decrease of the guanine oxidation peak current. The changes in the experimental parameters such as the accumulation time and the concentration of GMF were studied. Linearity of the guanine oxidation signals was obtained in response to the range of 0.5 – 8.0 μg/mL GMF. Detection and quantification limits in the range of μg/mL were determined.