Reactive oxygen species (ROS) play a dual role in the chemotherapy of cancer with cisplatin, providing both anti-tumor effects and contributing to drug resistance at various stages of treatment which seriously affects treatment effectiveness. The detailed mechanism of ROS is urgently necessary to be explored. To address this issue in the non-small-cell lung cancer (NSCLC) with cisplatin-resistance, a reliable assay was developed by synthesizing and characterizing an interesting near-infrared (NIR) ONOO^- probe BPB with high specificity, quick response (<30 s) and excellent limitation of detection (59 nM), which was further convinced through living cell imaging techniques providing different fluorescence variation between cell and cuvette. All the results revealed that ONOO^- might be a practical biomarker to comprehend the detail molecular mechanism in cisplatin-resistant A549 cells. The elevated levels of ONOO^- in cisplatin-resistant A549 cells, accompanied by a significant reduction in fluorescence following the knockout of miR-125a-5p in these cells and strong fluorescence without knockout of miR-125a-5p ignoring the presence of cisplatin. Comparing with cisplatin-resistant cells, the control would provide a rational background and then showed distinct fluorescence from BPB with ONOO^- toward cisplatin. This assay offers a promising tool for exploring the molecular mechanisms associated with miR-125a-5p and its potential linkage to therapeutic efficacy involving ONOO^- signaling. By utilizing this innovative assay, researchers can gain valuable insights into the treatment strategies and underlying mechanisms of cisplatin-resistant cancers, which should be beneficial to the therapy of cancers.