Mouse hepatitis virus (MHV) is a model coronavirus, but the traditional etiological methods are limited to the viruses isolated from nature. The reverse genetic system can rapidly obtain the recombinant virus for research, creating a new method to study the genomic functions of MHV and other coronaviruses. In this study, whole genome of the original MHV A59 strain was divided into six fragments by reverse transcription-polymerase chain reaction (RT-PCR). All fragments were individually cloned into plasmid vectors, then the full-length genomic RNA was obtained by restriction enzyme digestion, in vitro ligation and in vitro transcription. After RNA was electroporated into cells, the recombinant virus was rescued. The results revealed that the replication characteristics of recombinant virus rA59 is similar to the original strain, providing an experimental tool for studying the biological characteristics of the virus. To detect the virus replication level, the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc were obtained by inserting the green fluorescent protein ZsGreen (Zoanthus sp. green fluorescent protein) and luciferase Nluc (NanoLuc luciferase) into the open reading frame 4 (ORF4) genome, respectively. The results indicate that the reporter genes do not compromise viral replication and the reporter viruses can well reflect the viral replication property of their parental recombinant virus. These two viruses were used to verify the antiviral activity of remdesivir, by detecting the fluorescence intensity or luciferase activity after treatment. The results demonstrate that the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc are suitable for high-throughput drug screening and have promising potential for application. In summary, the successful establishment of the 6-plasmid-based reverse genetic system for MHV strain A59 provides a powerful tool for studying the biological characteristics of MHV and testing antiviral drugs.