Proximity induced intramolecular nucleotide strand displacement, which can be simply performed in a single tube or in a complex cellular environment, is one of the key mechanisms for the detection of biological targets, especially for significant genetic molecules. The host factor for RNA phage Qb replication (Hfq), with two distinct single stranded RNA binding sites, has excellent properties as an affinity ligand in a proximity induced reaction. In this research, a versatile RNA chaperone-Hfq assisted RNA annealing strategy for the sensitive detection of the intermediate product, oligouridylated RNA, in a genetic regulation process was developed. Benefiting from the high binding affinity of Hfq for the probe and the target, the sensitive determination of oligouridylated RNA in cell lysis and human cervical cancer (HeLa) cells was successfully achieved. This study has also revealed that the Hfq assisted RNA annealing strategy can be further extended and applied in specific microRNA analysis, and RNA related tumorigenicity and disease diagnosis.