Lung cancer is the leading cause of cancer-related death around the world. The circulating tumor DNA (ctDNA) of EGFR L858R in plasma is crucial for development, targeted drug therapy, and prognosis of non-small cell lung cancer, the main type of lung cancer. Accurately detecting ctDNA using conventional methods is challenging due to its characteristics, such as considerably short size, extremely low level, and short half-life. Thus, developing a rapid, accurate, and cost-effective method for ctDNA EGFR L858R detection is urgently needed. Herein, we developed an electrochemical biosensor of ctDNA EGFR L858R based on the CRISPR/Cas12a system and MB/ Fe3O4@COF/PdAu nanocomposites. The CRISPR/Cas12a system played roles in the precise recognition of ctDNA targets and indistinguishable cleavage of single-stranded DNA. Additionally, the MB/Fe3O4@COF/PdAu nano composite has good catalytic activity and signal amplification performance. The proposed electrochemical biosensor showed high specificity, stability, and selectivity. Notably, the limit of detection of the proposed biosensor was 3.3 aM. The detection results of 25 clinical samples showed that 22 and 20 positive samples were detected by electrochemical detection and droplet digital polymerase chain reaction, respectively. Therefore, we established a high-precision, reliable, and convenient method for ctDNA detection, which has a potential application in the diagnosis and prognosis of cancer.