RNA binding proteins provide a key layer of post-transcriptional gene regulation and determine RNA fate throughout the RNA life cycle. Previously, UV crosslinking and oligo-dT capture were employed to identify the repertoire of RBPs in different cellular contexts. Yet, such a method failed to capture an important class of RBPs that recognize long double-stranded RNAs (dsRNAs). Recently, aberrant immune activation by endogenous dsRNAs is receiving increasing attention as these RNAs are associated with various human degenerative and inflammatory diseases. However, our understanding of dsRNA regulation is lacking as only a handful of dsRNA-binding proteins (dsRBPs) are known. Here, we utilize dsRNA capture followed by quantitative mass spectrometry analysis for comprehensive identification of dsRBPs in proliferating human kidney epithelial cells. We find over 90 proteins that qualify as dsRNA-binding and further validate their interaction with in vitro synthesized dsRNAs. Notably, many of these RBPs are associated with viral replication, consistent with the abundant viral dsRNAs expressed in infected cells. We further perform CRISPR knockout screening to investigate the regulatory potential of dsRBPs and find a number of dsRBPs that can affect the downstream interferon response by exogenous dsRNAs. We find that depletion of two of these dsRBPs affected the replication of human beta-coronavirus OC43 and influenza A virus PR8. Our dsRNA interactome capture provides an unbiased and comprehensive characterization of dsRBPs and will facilitate our understanding of dsRNA regulation in physiological and disease contexts.