Lead ion is very harmful to the environment, so it is very important to study its detection methods. In this study, a novel electrochemical sensor was constructed by modifying deoxyribonucleic acid (DNA) on the electrode, which can be used for the detection of Pb2+ in the environment. Part of the mixed solution of chitosan (CS) and Pb2+ template ions was dropped onto the surface of a glassy carbon electrode. CS-Pb2+ film was cross-linked through sodium tripolyphosphate. And a novel DNA-imprinted sensor was prepared by electrodepositing CS-Pb2+ thin film with gold nanoparticles (AuNPs), removing Pb2+ templates, and immobilizing specific double-stranded DNA. The electroactive area, surface morphology, sensitivity, and electrochemical reaction mechanism of the DNA-imprinted sensor were analyzed. The elementary reaction steps were studied through electrochemical reaction kinetics analysis. The experimental results indicate that the DNA-imprinted electrochemical biosensor can quantitatively detect Pb2+ in the range of 10–100 μM (R2 = 0.9935), and its detection limit is 6.5074 μM (3σ/slope). The sensitivity of the electrochemical biosensor is 1.55233 × 10−6 A/μM, and its active areas is 6.233 cm2. The desorption mechanism and adsorption mechanism have been explored through dynamic parameter analysis. The novel DNA imprinted electrochemical biosensor developed in this paper provides a robust method for detecting lead ions in solution. Additionally, it establishes a solid groundwork for detecting other metal ions.
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