Background <p>ROP is a leading cause of childhood blindness, with inflammation influencing its progression. Tear fluid serves as a non-invasive medium for assessing biomarkers.</p> Aim <p>This study examines key inflammatory cytokines in tear fluid to identify biomarkers for ROP progression and severity.</p> Methods <p>Eighty preterm infants were grouped by ROP severity (No, Mild, Moderate, Severe; <i>n</i> = 20 each). Tear fluid cytokine levels were measured via ELISA and compared using one-way ANOVA with Bonferroni-adjusted post hoc tests.</p> Results <p>The levels of IL-1, IL-6, and TNF-α were significantly different across ROP severity groups; higher concentrations in more severe stages of ROP/moderate and severe than those in no ROP and mild ROP. However, no significant differences were found among the groups regarding IL-10, IL-17, and IL-22. In pairwise comparisons, the levels of IL-1 and IL-6 in most of the ROP severity groups showed significant differences according to the Bonferroni post hoc analysis, while no significant differences were observed regarding IL-10, IL-17, and IL-22.</p> Conclusion <p>The study further reveals that increased levels of the pro-inflammatory cytokines IL-1, IL-6, and TNF-α in tear fluid correlate with the severity of ROP. These findings suggest that tear fluid inflammatory cytokines may be regarded as potential biomarkers in the diagnosis and follow-up of ROP. This non-invasive approach presents a new opportunity for monitoring the disease process and assisting in clinical decisions. These observations need further confirmation and studies on their clinical application.</p>