For long, double-stranded RNAs (dsRNAs) have been associated with viral infection, but accumulating number of studies report that vertebrate cells can produce endogenous dsRNAs from both retroelements and mitochondrial DNA. They form structure-specific interactions with various dsRNA-binding proteins (dsRBP) to regulate cellular signaling by activating the innate immune response . Recent studies show that dsRNAs are closely associated with several human diseases that feature aberrant immune activation and tissue degeneration. However, our understanding on the regulation of cellular dsRNAs and their biological function is hampered by an incomplete annotation of tissue-specific profiles of dsRNAs. Here, we combine experimental and computational approaches to define the endogenous dsRNA repertoire in multiple cell types, including monocyte, neuronal progenitor cells, and several cancer cell lines of different tissue origins. We conduct formaldehyde cross-linking and immunoprecipitation followed by high-throughput sequencing (fCLIP-seq) using anti-dsRNA J2 antibody to capture and profile dsRNAs longer than 40 bp. A combination of alignment algorithm strategy to map unique reads with dsRNA-producing features and to examine the read distribution over genomic features is employed to characterize and locate cellular dsRNAomes of each cell type. Our comprehensive data on human dsRNAome atlas across different cell lines will provide an important basis to identify the list of genes regulated by endogenous dsRNAs, and understand the functional interplay among them in a tissue-specific context.