Hydrogen sulfide (H2S) is a new endogenously generated gasotransmitter and has implicated in many physiologies and pathologies closely related to its intracellular and intercellular signaling transduction. Although many fluorescent probes have been exploited to track and quantify H2S in living systems, none of them could be used for monitoring intercellular transmission of H2S. Herein, we developed a cell surface specific H2S probe, 4-azido-6-sulfo-N-hexadecyl-1,8-naphthalimide, sodium salt (ASNHN-N3), trying to investigate the behaviors of extracellular release of H2S. ASNHN-N3 is week fluorescent and could react with H2S at 37 °C in pH 7.4 buffer solutions to form product ASNHN-NH2 with strong fluorescence (Φ = 0.22). Using ASNHN-N3 as H2S probe, excellent linear correlation versus the concentration of NaHS was obtained ranging from 0 to 10 μM and the detection limit was 0.75 μM. With the lipid anchor and the hydrophilic sulfonic group introduced into the 1,8-naphthalimide (a skeleton of two-photon fluorescent probe), the amphiphilic probe is located at the surface of living cells which can record H2S efflux from the cell diffusing across the plasma membrane in living cells and deep-tissue by using two-photon microscopy. Thus we present a new strategy for further studying the mechanism of signaling molecules in cell communication and signal pathways.