It is important to quantify multiple biomarkers in a single run due to the advantages of precious samples and diagnostic accuracy. Based on the distinguishability of two types of current signals from single particle electrochemical collision (SPEC), steptype current transients produced by Pt nanoparticles (PtNPs) catalyzed hydrazine oxidation and peak-type current transients produced by Ag nanoparticles (AgNPs) oxidation, a kind of multiplex immunoassay of target microRNAs (miRNA-21 and Let-7a) have been established during SPEC in a single run. When the single-stranded DNA (ssDNA1) that was perfectly complementary to miRNA-21 was coupled to the surface of PtNPs, the SPEC of PtNPs electrocatalysis was inhibited and the step-type current transients disappeared, while the single-stranded DNA (ssDNA2) that was perfectly complementary to Let-7a was coupled to the surface of AgNPs, the SPEC of AgNPs oxidation was inhibited, and the peak-type current transients disappeared, thus the signals were in the "off" state at this time. After that, miRNA-21 and Let-7a were added into solution, complementary base pairing disrupted the weak DNA−NP interaction and restored the electrocatalysis of PtNPs and the electrooxidation of AgNPs, and the step-type current signals and peak-type current signals were in the "on" state. Moreover, the frequencies from two different recovered signals (PtNPs catalysis and AgNPs oxidation) corresponded to the amount of added miRNA-21 and Let-7a, thus a multiplex immunoassay method for dual quantification of miRNA-21 and Let-7a in a single run was established.