Detecting H2S in living cells has attracted a lot of attention because H2S is one of the most important gaseous mediator for regulation of cellular signal transduction pathways which is related to many diseases such as Alzheimer’s disease, Down’s syndrome, diabetes and liver cirrhosis. Two-dimensional (2D) nanomaterials possess the highest surface-to-volume ratio and have exceptional optical and electrical properties which make them extremely prospective for sensors applications. Previously, our group demonstrated the assembly of 2D membrane-mimetic nanomaterials from lipid-like peptoids (sequence-defined poly-N-substituted glycines). Large flexible surface area and good stability make these 2D nanomambranes have great potential in sensing probes. Due to the large side-chain diversity of peptoids, the surface functions of the 2D nanomembranes could be tuned and achieved easily through side-chain chemistry. Normally, fluorescent probes for H2S have been developed based on specific chemical reactions by taking advantage of the reducing or nucleophilic properties of H2S. In this work, the dinitrophenyl ether group were introduced into 3 position of 1, 8-naphthalimide to give the two-photo fluorescent group (Nap-NI), which acted as the H2S reactive site. Compared to traditional fluorescent probes work with one photo microscopy, two-photon fluorescent probes reduced phototoxicity, increased specimen penetration, and negligible background fluorescence. The Nap-NI groups were modified on the peptoid sequences (Pep-Nap-NI) to achieve self-assembled 2D nanomembranes using as H2S sensor. Atomic force microscopy (AFM), Transmission electron microscopy (TEM) and X-ray diffraction (XRD) results demonstrate that the assembled structures are highly crystalline and is similar with the model we developed in previous work. The absorption and fluorescence titration experiments of peptoid 2D probes with H2S were recorded in aqueous solution with different dosage of H2S. The selectivity of peptoid 2D probes for H2S over reactive oxygen species (ROS), reactive nitrogen species (RNS) was also verified. We will also tested the ability of the peptoid 2D probes to be used to visualize and monitor the H2S in live cells in the following experiments. In consequence, we developed a fluorescent probe for imaging H2S based on 2D nanomembrane assembled from peptoids, which could rapidly react with H2S in aqueous solution. It provides a new promising 2D platform as biosensor for imaging some biomarkers in living cells.