Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, evoke current research interest increasingly due to their potential applications in therapeutics and sensors. One of the most attention-attracting is the lead-specific DNAzyme, which is composed of an enzyme strand 17E and a substrate strand 17S, addition of Pb²⁺ enables the DNAzyme to cleave its substrate. In this study, we took advantage of the unique optical properties of a water-soluble cationic polythiophene (PT) and designed a fluorometric sensing assay for the detection of Pb²⁺. A simple “mix-and-detect” approach enabled the detection of Pb²⁺ within 20 minutes due to the distinguishable optical properties of PT–dsDNA and PT–ssDNA. As low as 10 nM Pb²⁺ could be detected with a detection range from 10 nM to 100 μM via this method. Furthermore, this method was highly selective and only minimally perturbed by nonspecific metal ions. Since common steps such as modification and separation could be successfully avoided, this simple, sensitive, specific, and cost-effective approach showed great potential applications in environmental monitoring, waste management and clinical toxicology.