Due to the largely recognized utility of bioluminescence in many fields, a wide variety of luciferase-luciferin systems have been investigated in order to find the best-suited for a number of different applications. The collected knowledge has allowed the identification of a few necessary, or at least desirable, properties, such as bright luminescence, low background signal and small dimension of the enzyme that must exhibit structural stability at operating conditions. The NanoLuc-furimazine pair seems to meet all these requirements, but the mechanism of the reaction and the characteristics of the species responsible for the emission remain unknown. The aim of this study is to identify the luminescent product among the possible forms of oxidized furimazine and to understand how the chemical form and structure of the system, before and after the oxidation, are involved into the reaction mechanism and determine emission. To do this, we consider two possible forms of furimazine, the keto and the enol one, and test which of them is the most plausible candidate in the bioluminescence process on the basis of enzyme-substrate interactions from docking calculations. A similar procedure is repeated for three possible forms of the furimamide luminescent product, and their properties in the protein environment are then evaluated via QM/MM calculations. In contrast with previous indications, our simulations well support the involvement of the enol form of furimazine as reagent and point to the zwitterionic forms of furimamide as emissive species.