Chemicals used in leather processing can penetrate the human skin and respiratory system, causing neuroinflammation and pose health risks. This study addresses the lack of effective treatments for this condition by focusing on the design and synthesis of a novel dual-channel fluorescence probe HMDP. HMDP, equipped with coumarin as the fluorophore, quinolinium ion as its mitochondrial localization group, and diphenylphosphinoyl as the O2•− recognition site, demonstrated significant dual-channel fluorescence amplification at 570 and 714 nm. Incorporating two non-interfering fluorescent channels enabled cross-verification, enhancing accuracy. This research systematically assessed fluctuations in O2•− levels in a cell oxidative stress model induced by scrap leather using HMDP. The results underscored the considerable potential of this mitochondrial-targeted near-infrared fluorescent probe for monitoring scrap leather-induced cell oxidative stress. This research contributes valuable insights into the neuroinflammatory effects induced by leather processing chemicals, providing a foundation for potential therapeutic interventions to mitigate health risks associated with such exposures.