DNA helicases are essential to genomic stability by regulating DNA metabolisms and their loss-of-function mutations lead to genomic instability and predisposition to cancer. Paradoxically, overexpression of DNA helicases is observed in several cancers. Here we analyzed genomic and molecular alterations in 12 important DNA helicases in TCGA pan-cancers to provide an overview of their aberrations. Significant expression heterogeneity of 12 DNA helicases was observed. We calculated DNA helicase score (DHS) based on their expression, and categorized tumors into high, low and intermediate subtypes. High DHS subtypes were robustly associated with stemness, proliferation, hyperactivated oncogenic signaling, longer telomeres, total mutation burden, copy number alterations (CNAs) and shorter survival. Importantly, tumors with high DHSs exhibited stronger expression of alternative end-join (alt-EJ) factors, indicative of sensitivity to chemo- and radio-therapies. High DHSs were also associated with homologous recombination deficiency (HRD), BRCA1/2 mutations and sensitivity to PARP inhibitors. Moreover, several drugs are identified to inhibit DNA helicases, with the Auror A kinase inhibitor Danusertib as the strongest candidate that was confirmed experimentally. The aberrant expression of DNA helicases was associated with CNAs, DNA methylation and m6A regulators. Our findings thus reveal widespread dysregulation of DNA helicases and their broad connection with featured oncogenic aberrations across human cancers. The close association of DHS with the alt-EJ pathway and HRD, and identification of Danusertib as a putative DNA helicase inhibitor have translational significance. Taken together, these findings will contribute to DNA helicase-based cancer therapy.