We used electroactive label crystal violet (CVi) as a G-quadruplex (G4) DNA intercalator for sensitive electrochemical sensing of Tl+. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) techniques were applied for electrochemical determination of Tl+ at the surface of carbon paste electrode as working electrode. Under optimized conditions, the difference in the DPV reduction peak currents of CVi before and after its intercalation into the formed G4 (ΔI) used an analytical signal for determination of Tl+ was linearly related to the concentration of Tl+ over the ranges of 0.4–50.0 nM and 0.1–10 μM. Limit of detection was calculated 0.341 nM (S/N = 3). The biosensor showed good selectivity and satisfactory results in real sample analysis of Tl+.