Glutathione (GSH) is an important biological thiol in cells, which is involved in many physiological processes in the organism and regulates pathological processes of cells. Rapid and accurate monitoring of GSH in vitro and in vivo is quite needed in investigating important biochemical events. In this contribution, innovative cerium (Ce) doped polyaniline (Ce-Fe@PANI NPs) were prepared via Fe(III) induced oxidization polymerization method. Upon addition of GSH, the absorption of Ce-Fe@PANI NPs red shifted from the visible to the NIR region, confirming the excellent absorption response to GSH. Moreover, Ce-Fe@PANI NPs exhibited excellent photoacoustic (PA) imaging enhancement in tube and shifted the PA intensity peak from 680 nm to 820 nm upon addition of GSH. In vitro and in vivo experiment verified that Ce-Fe@PANI NPs can monitor GSH in deep tissues via PA imaging technology. Collectively, this research provides Ce-Fe@PANI NPs would serve as a powerful nanoplatform to realize PA imaging detection of GSH in vitro and in vivo.