Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a very small sample volume, which is critical for non‐invasive early diagnosis of cancer. Multiplexing with nanopores: A new strategy for sensing multiple cancer biomarkers in a single sample through the combination of a sandwich assay and barcode DNA‐assisted nanopore sensing is reported. The characteristic events produced by each barcode DNA can be used to detect the presence of its corresponding biomarker and quantify its concentration. This strategy could lead to rapid and specific cancer diagnosis in a single drop of blood.