Multiplexing methods which are capable of measurement of multiple analytes in a single assay are of great importance in many fields. The conventional strategy for simultaneous detection of multiple species is to construct a sensor array. Herein, we report an innovative multiplex multi-analyte detection platform in a non-array format for protease measurement. By monitoring protease degradation of a single peptide substrate containing two cleavage sites for a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 10 (ADAM17) in a single nanopore, simultaneous detection and quantification of these two model proteases in mixture samples could satisfactorily be accomplished. Our developed multiplexing sensing platform has the potential to be coupled with the traditional sensor array to further improve the multiplexing capability of the sensor, which may find useful applications in clinical diagnosis and prognosis.