In summary, the fluorescence spectral tuning mechanism of bacterial luciferase has been systematically investigated from a theoretical perspective. In line with previous studies, the geometry confinement by the indirect steric effect of the protein surroundings is key to its fluorescence efficiency, as it maintains the chromophore in a planar conformation and blocks excited state deactivation via a CI-mediated internal conversion. On the other hand, the fine-tuning of the fluorescence spectroscopic properties, including emission wavelength and intensity, can be achieved through electrostatic effect, such as embedding the chromophore in a homogeneous dielectric environment or applying OEEFs. Increasing solvent polarity leads to a red-shift of the fluorescence emission maximum λ F and an increase in intensity. Furthermore, the effect of solvent polarity can be divided into two regimes, a low-polarity regime (ε < 25) and a high polarity regime (ε > 25). Both emission wavelength and intensity are more sensitive in the low-polarity regime than in the high-polarity regime. A red-shift in wavelength can also be achieved by applying a negative F X field along the long axis of the isoalloxazine ring. The emission maxima can be redshifted by more than 100 nm with a moderate filed strength of 0.0075 a.u. (38.6 MV/cm). Intriguingly, the redshift of the wavelength under F X is accompanied by a negligible change of intensity, in sharp contrast with the case of solvent polarity. OEEFs provide a more flexible method to control the fluorescence wavelength, a positive field blueshifting the emission wavelength.