A zero-background electrochemical DNA sensor is badly desired for highly accurate real-sample assay. Herein, we built a lambda exonuclease (λ-Exo)-assisted ligase chain reaction (LCR)-based electrochemical biosensor, named λ-eLCR, for highly sensitive CYP2C19*2 allele genotyping with zero-background by using the methylene blue (MB)-attached DNA probe and an inverted hybridization format. Specifically, the ligated MB-labeled single-stranded DNAs (ssDNA) were generated exponentially by LCR amplification followed by λ-Exo digestion in the presence of target DNA, then they were invertedly hybridized with the upper sequence of capture probes on the gold electrode, bringing the redox reporter-MB close enough to the surface of the electrode. In this regard, the MB-derived signal detected by square wave voltammetry technique was quantitatively related with the target DNA concentration and guaranteed the zero background. Under the optimum condition, the proposed λ-eLCR exhibited excellent performance in terms of good linear range (100 fM–10 pM) and a low limit of detection (100 fM) for CYP2C19*2 gene. Finally, this λ-eLCR successfully distinguished the CYP2C19*2 allele genotypes in human whole blood samples, which has a great prospect in individualized medicine.