Herein, we introduce a unique approach for discrimination between two important biothiols (BTs): cysteine (Cys) and glutathione (Glu), on the basis of changes in chromaticity coordinates of a dual-emitting "a few particle" nanoprobe. A novel dual-channel emission nanocomposite was developed by zinc-mediated conjugation of green-emitting fluorescein with red-emitting gold nanoclusters (Au NCs), hereafter referred to as Zn−FL−Au NCs. Cys and Glu having different interactions with Zn−FL−Au NCs were found to have different effects on the overall luminescence of Zn−FL−Au NCs, which eventually led to observation of different chromaticity indices of the latter upon interaction with these two BTs. For example, upon addition of Cys to a dispersion of Zn−FL−Au NCs having chromaticity coordinates (0.38, 0.49), the luminescence peak due to Au NCs was found to have been selectively quenched, as a consequence of which the chromaticity coordinates of Zn−FL−Au NCs changed to (0.33, 0.52). On the other hand, upon interaction with Glu, the intensity of the luminescence peak due to Au clusters as well as fluorescein (FL) enhanced to a certain extent, however, keeping the chromaticity coordinates almost similar to those of Zn−FL−Au NCs (0.38, 0.47). Allied results were obtained from super-resolution microscopic analysis wherein luminescence of "a few particle" of Zn−FL−Au NCs was used to probe the differential interaction with the aforementioned biothiols, leading to a different degree of changes in the Commission Internationale d'E ́clairage (CIE) coordinates of the former. Also, the specificity of the nanoprobe toward Cys was confirmed by monitoring the interactions of Zn−FL−Au NCs with other interfering chemical compounds. Moreover, the optical properties of Zn−FL−Au NCs were found to be retained following incubation in human blood serum, which further supports the use of this nanoprobe for discrimination of BTs in practical systems.