The development of novel biomarkers for early-stage diagnosis of prostate cancer (PCa) has attracted the attention of researchers in the last decade. Nucleolin (NCL) has emerged as a possible biomarker of PCa due to its high expression levels in the surface of PCa cells and affinity towards parallel G4s since it contains four RNA-binding domains (RBDs). Herein, we developed a novel strategy based on a microfluidic platform for the detection of NCL in biological samples, such as human plasma. The RNA G4 (rG4) sequence found in human precursor microRNA 92b (pre-miR-92b) was used as a molecular recognition probe since it forms a single dominant parallel rG4 conformation in the presence of 0.1 mM K as confirmed by NMR spectroscopy. The additional stability of the rG4 structure was provided by the acridine orange derivative ligand C, which stabilizes the pre-miR-92b rG4 structure, as denoted by an increase in more than 30 °C of its melting temperature. FRET-melting assay revealed a remarkable synergistic effect of NCL RBD1,2 and C on the stabilization of the pre-miR-92b rG4. The binding of pre-miR-92b to NCL RBD1,2 was determined by in silico studies, which revealed a binding pocket formed by a 12-residue linker between RBD1 and RBD2. Both, pre-miR-92b rG4 and pre-miR-92b rG4/C complex demonstrated high affinity towards NCL RBD1,2, as proved by fluorimetric titrations (K range between 10 and 10 M). The stability and nuclease resistance of pre-miR-92b rG4 and pre-miR-92b rG4/C complex were evaluated as molecular recognition probes to capture and detect NCL. Finally, the microfluidic platform detects NCL in complex biological samples, such as human plasma. Overall, this work demonstrates the usefulness of the microfluidic platform based on the pre-miR-92b to detect NCL and the possibility to be used as a valuable biomedical tool in PCa diagnosis.Copyright © 2020 Elsevier Inc. All rights reserved.