Breakthroughs in circulating tumor DNA (ctDNA) analysis are critical in tumor liquid biopsies but remain a technical challenge due to the double-stranded structure, extremely low abundance, and short half-life of ctDNA. Here, we report an electrochemical CRISPR/dCas9 sensor (E-dCas9) for sensitive and specific detection of ctDNA at a single-nucleotide resolution. The E-dCas9 design harnesses the specific capture and unzipping of target ctDNA by dCas9 to introduce a complementary reporter probe for specific molecular assembly and signal amplification. By efficient homogeneous assembly and interfacial click reaction, the assay demonstrates superior sensitivity (up to 2.86 fM) in detecting single-base mutant ctDNA and a broad dynamic range spanning 6 orders of magnitude. The sensor is also capable of measuring 10 fg/μL of a mutated target in excess of wild-type ones (1 ng/μL), equivalent to probing 0.001% of the mutation relative to the wild type. In addition, our sensor can monitor the dynamic expression of cellular genomic DNA and allows accurate analysis of blood samples from patients with nonsmall cell lung cancer, suggesting the potential of E-dCas9 as a promising tool in ctDNA-based cancer diagnosis.