Multifunctional enzymatic nanomaterials (nanozymes) with good biocompatibility, strong enzymatic activity, and substrate specificity are the desired alternatives to natural enzymes. Here, the design of a peroxidase mimetic nanozyme capable of detecting and depleting glutathione (GSH) in the bacterial cells is presented. The nanozyme palladium-iron nanostructure decorated graphdiyne nanosheet (PdFe/GDY) was prepared by a facile hydrothermal approach. It can efficiently catalyze the decomposition of H₂O₂ to produce •OH by a peroxidase-like mechanism. Surprisingly, the PdFe/GDY nanozymes can detect GSH in bacteria with excellent specificity and ultra-low detection limit of 24.45 nM. Moreover, the PdFe/GDY nanozymes can consume GSH for bacterial disinfection with killing efficacy >99.99%, promote wound healing and do not cause significant toxicity in vitro and in vivo. To our knowledge, this is the first example of a GDY-based nanozyme biosensor and antibacterial agent. Collectively, this work would be indicative to construct GDY-based nanozymes in clinical diagnosis and sterilization.