Autoimmune thyroid diseases (AITD) often interfere with early detection due to asymptomatic symptoms and normal thyroid function in routine tests. Developing early diagnostic tools is crucial for timely and accurate treatment, potentially reducing complications and improving patient outcomes. In this study, we developed Ox-NE, a novel fluorescent probe designed for the specific detection and quantification of neutrophil elastase (NE), a key biomarker of inflammation. Unlike the traditional probes, Ox-NE utilizes a unique mechanism that minimizes background fluorescence and enhances photostability, offering rapid, non-invasive, and apparent diagnostic capabilities. Ox-NE had a low limit of detection (LOD) of 1.54μg/mL and exhibited high sensitivity and specificity with strong anti-interference properties. Through in vitro experiments, Ox-NE could accurately detect elevated NE levels in the serum of thyroiditis patients. Additionally, it could differentiate between normal thyroid cells, inflamed thyroid cells, and thyroid cancer cells. It also effectively facilitated the screening of sivelestat, a therapeutic agent for thyroiditis. Successful fluorescence imaging in mouse models further confirmed the potential of Ox-NE in advancing thyroid disease diagnostics.