The development of a highly selective and sensitive Ba2+ sensor is crucial because of its industrial, environmental, and biological relevance. This study introduces a novel coated wire barium-selective electrode incorporating DNA as an ecofriendly natural ionophore in a plastic membrane, utilizing dioctyl phthalate as a plasticizer. The use of DNA as an ionophore provides enhanced selectivity and sensitivity, showcasing a slope of 33.15 mV/decade across a broad concentration range (1 × 10⁻5 to 1 × 10⁻2 M). The sensor exhibited a rapid response time of 9 s, a wide pH tolerance (2.6–6.9), and good selectivity for Ba2+ over other cations. Characterization of the membrane using FT-IR, SEM, and EDX confirmed its structural and morphological features. Practical applicability was demonstrated by detecting Ba2+ in spiked samples (milk, juice, tap water and urine) with recovery rates of 96.07–98.9%. This DNA-based approach offers a promising advancement in ion-selective electrode technology, with significant implications for real-world Ba2+ detection.