Fluorogenic bioorthogonal probes are ideal for fluorescent imaging in live cell conditions. By taking advantage of the dual functionality of tetrazine (Tz), as a bioorthogonal reaction unit as well as a fluorescence quencher, a fluorophore−Tz conjugate (FLTz) has been utilized for fluorescent live cell imaging via inverse electron-demand Diels−Alder (iEDDA) type bioorthogonal reactions. However, most FLTz strategies rely on a donor−acceptor type energy transfer mechanism, which limits red-shifting of probes’ emission wavelength without deterioration of the fluorescent turn on/ off ratio. To address this constraint, we present a monochromophoric design strategy for making a series of FLTzs spanning a broad range of emission colors. For the systematic comparison of design strategies with minimized structural differences, we selected indolizine-based emission-tunable Seoul-Fluor (SF) as a model fluorophore system. As a result, by inducing strong electronic coupling between Tz and π-conjugation systems of an indolizine core, we efficiently quench the fluorescence of SF−tetrazine conjugates (SFTzs) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cyclooctene (TCO). Importantly, we were able to develop a series of colorful SFTzs with a similar turn-on/off ratio regardless of their emission wavelength. The applicability as bioorthogonal probes was demonstrated with fluorescence bioimaging of innate microtubule and mitochondria using docetaxel−TCO and triphenylphosphonium−TCO in live cells without washing steps.