Enterovirus 71 (EV71), belonging to the Picornaviridae family, presents a significant concern as it is a causative agent of hand-foot-and-mouth disease (HFMD), predominantly affecting small children. The potential for severe neurological complications, leading to mortality rates of up to 19%, underscores the urgency for research endeavours to counter this pathogen1. With escalating outbreaks in the Asia-Pacific region, there is a pressing need to explore novel therapeutic approaches. While current vaccines target specific serotypes, their efficacy lacks evidence of cross-protection. This prompts investigation into targeted antiviral therapies1,2. However, this pursuit is hindered by gaps in understanding EV71's replication cycle. The core objective of this study is to explore uncharted aspects of enterovirus RNA replication, with a particular emphasis on its interaction with genome packaging and viral assembly. A pivotal aspect of unravelling the molecular intricacies of the replication process involves the development of a fluorescent reporter system to visualise viral replication sites. This poses a challenge as direct modification of the viral genome is risky since it may affect the viral life cycle or infectivity. Hence, here we present the establishment of a fluorescent reporter system which will enable the specific labelling of viral replication sites without altering the viral genome. The system will serve as a crucial tool in guiding subsequent research phases, including correlative light and electron microscopy for precise lamella preparation and cryo-electron tomography data collection.