miRNA-21-3p is overexpressed in a number of cancers and contributes to their development with a concomitant inhibition of the p53 onco-suppressive function. While a direct interaction of p53 with some miRNA precursors (namely pri-miRNAs and pre-miRNAs) was found, no interaction with mature micro RNA has been so far evidenced. It could therefore be very interesting to investigate if a direct interaction of miR-21-3p and p53 is occurring with possible impairment of the p53 onco-suppressive function. Fluorescence and Atomic Force Spectroscopy (AFS) were applied to study the interaction of p53 DNA Binding Domain (DBD) and miRNA-21-3p. Förster resonance energy transfer (FRET) was used to measure the distance between the DBD lone tryptophan (FRET donor) and a dye (FRET acceptor) bound to miRNA-21-3p. AFS and Fluorescence evidenced a direct interaction between miRNA-21-3p and DBD; with the formed complex being characterized by an affinity of 105 M, with a lifetime in the order of seconds. FRET allowed to determine an average distance of 4.0 nm between the DBD lone Trp146 and miRNA-21-3p; consistently with the involvement of the DBD L3 loop and/or the H1 helix in the complex formation, directly involved in the oligomerization and DNA binding. This may suggest that a functional inhibition of p53 could arise from its interaction with the oncogenic miRNA. Evidence of DBD-miRNA-21-3p complex formation may deserve some interest for inspiring novel therapeutic strategies. Copyright © 2019 Elsevier Inc. All rights reserved.