Understanding the iris colour might be a useful biomarker in predicting emotional health status. We aimed to examine the relationship of iris colour with subjective emotional status in a sample of Chinese adolescents aged 13 to 14 years.A total of 2346 students from ten middle schools in Mojiang participated the examination. The emotional health status was measured by the adolescent self-reported PedsQL 4.0. We obtained standardized slit-lamp photographs and developed a grading system assessing iris colour (higher grade denoting darker). Logistic regression models were fitted to assess the relationship between iris colour and emotional status.The trend of decreasing negative emotional status with darker iris colour was significantly. Compared with grades 1 and 2 of iris colour, the students with grades 4 and 5 had less negative emotional status (ORfear = 0.56, 95 % CI: 0.41, 0.76; ORsadness = 0.67, 95 % CI: 0.49, 0.92; ORanger = 0.71, 95 % CI: 0.52, 0.98). In addition, the trend did not change after adjusting for potential confounders, including height, waist circumstance, sleep habits and computer times in logistic regression models.Only negative emotional indexes were selected as evaluative materials, it is unclear whether the pattern found in the present study also exists with regard to positive emotional indexes, such as happiness.Darker iris colour might be associated with less negative emotional status in Chinese adolescents.Copyright © 2023. Published by Elsevier B.V.