Freshness of fish is of importance for consumers because of its connection with health and taste. A mixture of complex volatile biogenic amines collectively known as total volatile basic nitrogen (TVB-N) are released from the fish spoilage due to enzyme-catalyzed decomposition of biogenic amines by spoilage microorganisms. The release of TVB-N, due to microorganism growth in fish samples are considered as potential analytical indicator of fish spoilage. The developed sensor contains a mixture of pH sensitive anionic dye, cresol red (CR) and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) above the critical micellar concentration. The mixture of CR-CTAB (1:1) was immobilized on the reduced graphene oxide (rGO) modified electrode surface. A change in static voltage was observed across the CR-CTAB/rGO biosensor when TVB-N are released from spoiled fish under room temperature. The developed biosensor can be used for the real time measurement of fish freshness.