A polarity-sensitive fluorescence probe AMN was developed to demonstrate the role of autophagy inhibitory drugs in the process of leather residue-induced neuroinflammation, promoting the knowledge of the relationship between autophagy and neuroinflammation. AMN showed a turn-on fluorescent signal in the process of autophagy inhibition via two-photon confocal imaging, which is different from the current popular autophagy probes. Therefore, AMN can offer high-sensitive imaging analysis of the autophagy inhibition process to better understand the role of autophagy in the process of neuroinflammation. The model of scrap leather-induced neuroinflammation using PC12 cells demonstrated that neuroinflammation can induce autophagy by releasing reactive oxygen species (ROS), and autophagy can alleviate neuroinflammation significantly via ROS scavenging.