Herein, we introduce a novel assay for multiple-gene recognition by ligation–double transcription mediated fluorometric profiling. We demonstrated the capability of the system to identify potential multi-gene classifiers for diagnostic use by employing a combination of a ligation–double transcription approach with a selective fluorophore probe–RNA hybridization/graphene oxide quenching system. The system is efficient and requires only 45 minutes for total experimentation and offers high sensitivity (369.6, 408, and 407.8 copies per mL for the O, E, and N genes of SARS-CoV-2, respectively) and specificity (selective to until two mismatches). Our system is expected to expedite the precise diagnosis of RNA-virus-related diseases with multiple gene classifiers. By focusing on distinct viral genes, our method allowed for the detection of various RNA viruses in a variety of sample pools.