Palladium(0) as one of the vital transition metals, is employed in numerous industries, such as drug synthesis, aerospace high-tech field and automobile industry. When the Pd(0) enter into the body, it will bind with thiol-containing amino acids, DNA, RNA, and other biomolecules damaging to human health. Thus, developing a novel tool for monitoring and imaging of Pd(0) in vivo is very urgent. In the work, based on a intramolecular charge transfer (ICT) mechanism a two-photon fluorescent probe NIPd had been designed and synthesized for the recognition Pd(0). In vitro experiments data displayed that probe NIPd exhibited a 13-fold fluorescent increase for Pd(0) in 30 min in the aqueous solution with a detection limit of 16 nmol/L. It also showed the outstanding selectivity and antijamming performance. More importantly, NIPd could be served as a two-photon fluorescent probe for real-time monitoring Pd(0) in living cells and mice.