The present paper aims to evaluate the stability of the recombinant coxsackeivirus B3 (CVB3) variant integrated with the open reading frame (ORF) of mCherry, a red fluorescent protein gene. Recombinant virus CVB3-mCherry was recovered by transfecting HeLa cells with pCVB3-mCherry. The virulence of the recombinant virus was determined by plaque forming unit (PFU) assay, and the expression of mCherry in the infected HeLa cells was observed in fluorescence microscopy. The existence of the inserted mCherry sequence in CVB3-mCherry extracted from a serial of passages in HeLa cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. The results showed that both cytopathic effect and mCherry expression was observed in HeLa cells transfected with pCVB3-mCherry. Destablization of CVB3-mCherry genome was observed as early as in the second passage. The inserted mCherry gene as well as part of the CVB3 genome sequence was deleted which cause lethal mutations of the virus due to the shift or partial deletions of viral ORF. In conclusion, recombinant CVB3-mCherry could be used to study the infection of CVB3, while the recombinant construction showed an increased instability upon passages due to the insertion of mCherry ORF. Therefore, for the application of CVB3-mCherry, less than two passages of CVB3-mCherry are desirable and recovering the virus from the recombinant plasmid is recommended if longer period of application is considered.