Hydrogen sulfide (HS) serves an effective role in biological systems as the acknowledged third endogenous gasotransmitter, so it makes great sense to detect and analyze HS sensitively and quantitatively in subcellular environments, such as in mitochondria and lysosomes where HS is widespread and functions as the mediator. Considering the excellent photophysical properties and multiple modification sites, N-annulated perylene (NP) was firstly chosen as the fluorophore to design a series of colorimetric and ratiometric near-infrared (NIR) fluorescent probes for the sensitive and selective detection of HS. The probes showed near-infrared fluorescence at 681 nm in the absence of HS. But with the addition of HS, the NIR fluorescence decreased sharply and a new fluorescence peak at approximately 481 nm dramatically increased in a short response time, which could be clearly observed using the naked eye. Their large ratiometric fluorescence changes (about 200 nm), excellent selectivity and stability would be helpful for its detection in biological systems, and the limit of detection of the probe was calculated down to 139 nM. The reaction mechanism was studied as well. The targetable probes (Mito-NPNM and Lyso-NPNM) were also successfully employed to detect endogenous HS in the mitochondria and lysosomes of living cells respectively. Besides, these probes were successfully applied to quantify HS at low concentrations in serum where HS levels are of great significance as an important indicator of various diseases.