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Abstract Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications ‐ bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we usedGaussialuciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV‐1 DNA and IFN‐γ. We demonstrated that the incubation of Gluc conjugated HIV‐1 and IFN‐γ oligonucleotide probes with GO provided for monitoring of probe‐target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN‐γ and HIV‐1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN‐γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter.