A series of multistimuli-responsive [2]rotaxanes with a naphthalimide-functionalized macrocycle threaded through an axle containing a photochromic spiropyran (SP) or merocyanine (MC) stopper were fabricated and studied for their distinct photophysical properties in semiaqueous solutions. By different combinations of chemical and photochemical switchable stimuli, these [2]rotaxanes could be interconverted between multiple states, including monofluorophoric [2]rotaxanes with the close form of the SP unit, i.e., Rot-H-SP and Rot-SP before and after shuttling upon acid−base controls, respectively, along with their corresponding bifluorophoric [2]rotaxanes possessing the open form of the MC unit, i.e., Rot-H-MC and Rot-MC after UV exposure. Interestingly, the photoinduced electron-transfer (PET) effects appeared between nonemissive SP and aggregation-caused quenching (ACQ) naphthalimide units in [2]rotaxanes Rot-H-SP and Rot-SP, whereas the Förster resonance energy transfer (FRET)/Dexter energy transfer occurred in [2]rotaxanes Rot-H-MC and Rot-MC between green-emissive naphthalimide donor (λem = 528 nm) and red-emissive MC acceptor (λem = 648 nm) moieties after UV exposure. Moreover, the PET as well as FRET/ Dexter energy-transfer processes and the speculated molecular arrangements (with/without macrocyclic tilting) of all [2]rotaxanes and related derivatives were verified by time-resolved photoluminescence (TRPL) measurements and theoretical studies. Among all [2]rotaxanes and derivatives, Rot-MC in tetrahydrofuran (THF)/H2O (2:8, v/v) revealed the strongest red MC emission with the most effective FRET process that possessed attractive ratiometric photoluminescence (PL) due to the ideal shortest donor−acceptor distance. Regardless of pH and temperature effects, the highest red MC emissions with the optimum FRET processes of all compounds were maintained at room temperature in near-neutral conditions (i.e., pH = 5−9). Importantly, the red-emissive [2]rotaxane Rot-MC showed high selectivity and sensitivity toward sulfite-ion sensing to recover green donor emission via FRETOFF behavior owing to the Michael reaction of the MC moiety with the sulfite ion, which had an excellent limit of detection (LOD) value of 0.76 μM to be further utilized for the cellular imaging of sulfite detection in living cells. Accordingly, the novel ratiometric sensor approaches of bifluorophoric [2]rotaxane systems have been well developed in this study as the first rotaxane application of FRET processes toward sulfite detection with higher sensitivities than those of their monofluorophoric analogues. Various LOD values could be evaluated to realize the sulfite-sensing mechanisms of well-designed [2]rotaxane systems by the shuttling of macrocyclic fluorophores along with photoswitchable FRET behaviors via alternative UV−vis exposures.