Abstract: The bacterial RNA chaperone protein Hfq (Host factor for phage Qβ replication) is a conserved RNA-binding protein that serves as a central regulator of RNA-RNA interactions and is ubiquitously conserved across diverse bacterial species. A subset of small non-coding RNAs (sRNAs) in bacteria rely on Hfq to execute their post-transcriptional regulatory functions and Hfq facilitates the base-pairing complementarity between sRNAs and their target mRNAs, thereby modulating translational activity or mRNA stability. Although studies on Hfq have progressed much in recent years, its precise recognition mechanisms and dynamic regulatory processes remain obscure. Further elucidation of the molecular mechanisms of Hfq’s interactions with sRNAs and target mRNAs is needed. This review synthesizes current research advances to comprehensively delineate the structural characteristics of Hfq, its cooperative mechanisms with sRNAs, methodologies for capturing and identifying target RNAs, and its multifaceted biological roles, which facilitate understanding the regulatory network controlled by Hfq, especially in bacterial metabolism and virulence regulation.