Hydrogen polysulfide (HS, n > 1), a member of reactive sulfur species (RSS), is primarily generated during the crosstalk between HS and reactive oxygen species (ROS), which plays important role in physiological and pathological processes. Ferroptosis is a new non-classical mode of cell death, in which ROS-associated lipid peroxidation and iron-dependent accumulation are the main features. However, the biological effects of HS on ferroptosis and the detailed mechanisms of action remain poorly understood. Thus, there is an urgent need to develop highly selective and sensitive chemical tools for monitoring HS in living cells. Herein, we develop a two-photon fluorescent probe (PSP) for specifically imaging HS in live cells and tumor spheroids. This probe exhibited a sensitive and selective response to HS, which had been used for imaging exogenous and endogenous HS in living cells by confocal imaging and high content imaging. PSP exhibits excellent photo-stability and two-photon imaging performance when irradiating at 880 nm in 3D HeLa multicellular tumor spheroids. Importantly, our studies revealed that HS levels were significantly up-regulated during ferroptosis. These excellent properties ensure that PSP is a promising two-photon probe for exploring the biological and pathological effects of HS during ferroptosis.