Tear fluid, as an important ocular surface fluid, can effectively reflect both ocular and systemic metabolic states through its compositional changes, making it an ideal source for discovering disease biomarkers. Current tear collection methods mainly include the Schirmer strip test and microcapillary collection, while detection technologies encompass enzyme-linked immunosorbent assay, protein chip technology, mass spectrometry, Olink targeted proteomics, and bead-based multiplex assays. Studies have shown that various biomarkers in tear fluid—such as proteins, cytokines, and chemokines that are closely associated with the pathophysiological processes of fundus diseases including diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, retinopathy of prematurity, and uveitis, demonstrating potential as indicators for early diagnosis, disease assessment, and therapeutic monitoring. As a non-invasive and convenient detection tool, tear analysis shows broad application prospects in the diagnosis and treatment of fundus diseases. However, further optimization of collection and detection techniques, along with large-scale clinical studies to validate the clinical utility of tear biomarkers, is still needed to promote their standardization and widespread adoption in clinical practice.