Bioluminescence of bacteria is widely applied in biological imaging, environmental toxicant detection, and many other situations. Understanding the spectral tuning mechanism not only helps explain the diversity of colors observed in nature but also provides principles for bioengineering new color variants for practical applications. In this study, time-dependent density functional theory (TD-DFT) and quantum mechanics and molecular mechanics (QM/MM) calculations have been employed to understand the fluorescence spectral tuning mechanism of bacterial luciferase with a focus on the electrostatic effect. The spectrum can be tuned by both a homogeneous dielectric environment and oriented external electric fields (OEEFs). Increasing the solvent polarity leads to a redshift of the fluorescence emission maximum, λF, accompanied by a substantial increase in density. In contrast, applying an OEEF along the long axis of the isoalloxazine ring (X-axis) leads to a significant red- or blue-shift in λF, depending on the direction of the OEEF, yet with much smaller changes in intensity. The effect of polar solvents is directionless, and the red-shifts can be attributed to the larger dipole moment of the S1 state compared with that of the S0 state. However, the effect of OEEFs directly correlates with the difference dipole moment between the S1 and S0 states, which is directional and is determined by the charge redistribution upon deexcitation. Moreover, the electrostatic effect of bacterial luciferase is in line with the presence of an internal electric field (IEF) pointing in the negative X direction. Finally, the key residues that contribute to this IEF and strategies for modulating the spectrum through site-directed point mutations are discussed.