Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with limited therapeutic options. Here, we investigated the mechanisms of HCC in response to ROS1-targeted therapy.Recombinant RNases were purified, and the ligand-receptor relationship between RNase7 and ROS1 was validated in HCC cell lines by Duolink, immunofluorescence, and immunoprecipitation assays. Potential interacting residues between ROS1 and RNase7 were predicted by protein-protein docking approach. Oncogenic function of RNase7 was analyzed by cell proliferation, migration, invasion assays and xenograft mouse model. The anti-ROS1 inhibitor treatment efficacy was evaluated in HCC patient-derived xenograft (PDX) and orthotopic models. Two independent HCC patient cohorts were analyzed to evaluate the pathological relevance by immunohistochemistry. Plasma level of RNase7 in HCC patient was detected by ELISA.RNase7 associated with ROS1's N3-P2 domain and promoted ROS1-mediated oncogenic transformation. HCC patients exhibited elevated plasma RNase7 levels compared with normal subjects. High ROS1 and RNase7 expression were highly associated with poor prognosis in HCC. In both HCC PDX and orthotopic mouse models, ROS1 inhibitor treatment markedly suppressed RNase7-induced tumorigenesis and led to decreased plasma RNase7 level with tumor shrinkage in mice.RNase7 serves as a high-affinity ligand for ROS1, and plasma RNase7 has the potential to function as a biomarker to stratify HCC patients for anti-ROS1 treatment.