MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases. Detection and quantification of miRNAs is currently performed through complex and time consuming procedures. Herein we demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by Reflective Phantom Interface (RPI) label-free optical biosensor. We achieved sub-pM quantification of different miRNAs in about 1.5 h, through specific capture with surface DNA probes combined to a 35-fold mass amplification by an antibody targeting DNA-RNA hybrids and polyclonal secondary antibody, all performed without washing steps. The assay is the result of a modelling and optimization of the multi-step process that has been made possible by the RPI characterization of each individual interaction involved.