Update information. Double-stranded RNA (dsRNA) binding proteins (dsRBPs) play crucial roles in various cellular processes, especially in the innate immune response. Comprehensive characterization of dsRBPs is essential to understand the intricate mechanisms for dsRNA sensing and response. Traditional methods have predominantly relied on affinity purification, favoring the isolation of strong dsRNA binders. Here, we adopt the proteome integral solubility alteration (PISA) workflow for characterizing dsRBPs, resulting in the observation of 18 known dsRBPs and the identification of 200 potential dsRBPs. Next, we focus on zinc finger protein 385A (ZNF385A) and discover that its knockout activates the transcription of interferon-βin the absence of immunogenic stimuli. The knockout of ZNF385A elevates the level of endogenous dsRNAs, especially transcripts associated with retroelements, such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), and long terminal repeat (LTR). Moreover, loss of ZNF385A enhances the bioactivity of 5-Aza-2'-deoxycytidine (5-AZA-CdR) and tumor-killing effect of NK cells. Our findings greatly expand the dsRBP reservoir and contribute to the understanding of cellular dsRNA homeostasis.