A supramolecular host-guest [2]pseudo-rotaxane polymer containing blue-emissive (λem = 425 nm) anthracenebased di-topic donor guest (AN-G) and green-emissive (λem = 530 nm) napthalimide-based di-topic acceptor host (NP–H) after hydrogen sulfde (HS- anion) detection in semi-aqueous media was developed in this study. Towards HS- detection, the bi-fluorophoric [2]pseudo-rotaxane polymer possessed the best FRET process in the THF/Water solution (60% H2O, v/v), which was confrmed by TRPL measurements with the shortest lifetime of 1.1 ns and FRET effciency of 81.7% in comparison with the longer lifetime of 6.0 ns for mono-fluorophoric [2] pseudo-rotaxane polymer analogue before detection of HS- . The 1:1 host–guest molar ratio and the formation of the [2]pseudo-rotaxane polymer can be further verifed using 1H NMR complexation and DOSY experiments. The effects of pH and temperature were also investigated to fnd that an effcient FRET process of the host-guest could occur at lower and neutral pH values (i.e., pH = 1–9) and in the normal temperature range of 25–40 ◦C. As the napthalimide can react with HS- anion to induce green turn-on emission behavior, the napthalimide-based mono-fluorophoric guest could detect HS- anion by turn-on photoluminescence with a corresponding limit of detection (LOD) value of 1.04 μM, which was also confrmed to possess a very high selectivity of HS- detection compared with another 23 analytes, including 16 anionic species. In contrast, the optimum LOD value of 0.30 μM for bi-fluorophoric host-guest polymer as a FRET-ON sensor material towards HS- detection was achieved due to the ratiometric emission of distinct color change from blue donor to green acceptor. Therefore, host-guest [2] pseudo-rotaxane polymer can be employed for the future bio-imaging applications of HS- anion detection in living cells.