Objective To screen for the effective immunostimulatory sequences from hepatitis C virus(HCV) single strand RNA(ssRNA), and to identify the proteins that can bind to the sequences specifically. Methods Four oligonucleotide sequences (HCV-ORNs) derived from HCV ssRNA were synthesized, and the cytokines induced by them were detected using enzyme linked immunosorbent assay (ELISA), so as to identify the effective immunostimulatory sequences. Then proteins binding to the sequence(s) specifically were identified using improved electrophoresis mobility shift assay (EMSA) combined with electrospray ionization mass spectrum (ESI-MS). Results One of the HCV-ORNs, ORNHCVtail, induced higher levels of TNF-α and IFN-α (P<0.01). Totally 83 groups of proteins were identified that can bind to ORNHCVtail; the proteins included TLR3(Toll-like receptor 3), NLRC3(NLR family, CARD domain-containing 3), and NLRC4(NLR family, CARD domain-containing 4). Conclusion We have successfully screened out effective immunostimulatory sequence of HCV ssRNA and the proteins that can bind to the sequence specifically. Our findings in the present study lay a foundation for mechanism research of immunity stimulated by HCV infection.