Second near-infrared (NIR-II) window optical molecular imaging kicks off a new revolution in high-quality imaging in vivo, but always suffers from the hurdles of inevitable tissue autofluorescence background and NIR-II probe development. Here, we prepare a Förster resonance energy transfer-based ratiometric NIR-II window hydrogen sulfide (H ₂ S) sensor through the combination of an H ₂ S-responsive NIR-II cyanine dye (acceptor, LET-1055) and an H ₂ S-inert rhodamine hybrid polymethine dye (donor, Rh930). This sensor not only exhibits high sensitivity and selectivity, but also shows rapid reaction kinetics (~20 min) and relatively low limit of detection (~96 nM) toward H ₂ S, allowing in vivo ratiometric NIR-II fluorescence imaging of orthotopic liver and colon tumors and visualization of the drug-induced hepatic H ₂ S fluctuations. Our findings provide the potential for advancing the feasibility of NIR-II activity-based sensing for in vivo clinical diagnosis.