<p>Enzyme-linked immunosorbent assay (ELISA) has been widely used in cancer diagnostics due to its specificity, sensitivity and high throughput. However, conventional ELISA is semiquantitative and has an insufficiently low detection limit for applications requiring ultrahigh sensitivity. In this study, we developed an α-hemolysin-nanopore-based ELISA for detecting cancer biomarkers. After forming the immuno-sandwich complex, peptide probes carrying enzymatic cleavage sites are introduced, where they interact with enzymes conjugated to the detection antibodies within the complex. These probes generate distinct current signatures when translocated through the nanopore after enzymatic cleavage, enabling precise biomarker quantification. This approach offers a low detection limit of up to 0.03 fg ml^–1 and the simultaneous detection of six biomarkers, including antigen and antibody biomarkers in blood samples. Overall, the nanopore-based ELISA demonstrates high sensitivity and multiplexing capability, making it suitable for next-generation diagnostic and point-of-care testing applications.</p>