Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs.NS1 is known to bind non-specifically to dsRNAs as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs.Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5’terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide-sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5’-GUAAC / 3’-CUUAG double-stranded motif, which closely resembles the canonical 5’-splice site.Through an in depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA binding domain (RBD) specifically recognizes sequence patterns in a structuredependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar KD values) that leads to oligomerization of the RNA binding domain on its RNA ligands.