Abstract: The epidemic of Zika virus (ZIKV) raises critical public health and safety problems. However, there are currently no vaccines or drugs that are clinically approved for ZIKV infections. Since RNA-dependent RNA polymerase (RdRp) plays an important role in replication and transcription of ZIKV and is absent in human beings, it is a potential drug screening target of anti-ZIKV agents. According to the fluorescence-based alkaline phosphatase-coupled polymerase assay method, we established the NS5 RdRp inhibitor screening model. Through screening from an anti-infection compound library, we found a compound octenidine dihydrochloride (OCT) that could inhibit ZIKV RdRp activity with a half maximal inhibitory concentration (IC₅₀) of 5.43 μmol·L^-1. Biolayer interferometry (BLI) assay showed that OCT could bind to ZIKV RdRp and had a strong affinity. Moreover, OCT exhibited an inhibitory effect on ZIKV replication with a half maximal effective concentration (EC₅₀) of 29.94 μmol·L^-1. All these results indicated that OCT had the anti-ZIKV activity by targeting ZIKV RdRp, and it is likely to be a promising lead compound against ZIKV.