A multi-stimuli responsive pseudo [3]rotaxane NI-DBC/RB-DBA was composed of a green-emissive (λem = 530 nm) naphthalimide-based macrocyclic host and a turn-on red-emissive (λem = 595 nm) rhodamine-based guest towards Fe3+ ion, which illustrated interesting photo-physical properties in dioxane/H2O (4:6, v/v) solutions. Due to the chelation-induced ring unfolded form of red-emissive rhodamine unit upon the addition of Fe3+ ion, mono-fluorophoric pseudo [3]rotaxane NI-DBC/RB-DBA could be transformed into bi-fluorophoric pseudo [3] rotaxane NI-DBC/RB-DBA-Fe3+ to reveal the Forster ¨ resonance energy transfer (FRET) process, which could be further identifed by time-resolved photoluminescence (TRPL) measurements to obtain a higher energy transfer effciency of 45.4% than that (21.1%) of its analogous mixture without host-guest interactions. Regarding pH effects, both pseudo [3]rotaxane systems NI-DBC/RB-DBA and NI-DBC/RB-DBA-Fe3+ could also be employed as pH sensors in acidic (pH = 1–6) and basic (pH = 8–12) conditions, respectively. Moreover, the pH range of 6–8 could be appropriate to maintain excellent sensing capabilities of pseudo [3]rotaxane NI-DBC/RB-DBA to detect Fe3+ ion. Accordingly, pseudo [3]rotaxane NI-DBC/RB-DBA could show the FRET-ON sensing behaviour towards Fe3+ ion, which was attributed to the turn-on red-emissive rhodamine-functionalized RB-DBA acceptor. Owing to the best FRET process and ratiometric fluorescence of bi-fluorophoric pseudo [3]rotaxane NI-DBC/RB-DBA-Fe3+ after Fe3+ ion detection, pseudo [3]rotaxane NI-DBC/RB-DBA provided a prominent limit of detection (LOD) value of 39.01 nM, which was further proceeded as simple test strips towards Fe3+ ion recognition and applied to the detection of Fe3+ ion in various pristine water samples. Therefore, the developed pseudo [3]rotaxane with host-guest interactions has been well designed and constructed to utilize FRET-ON processes as a novel Fe3+ ion sensor.