Background <p>This study explored the correlation between hemoglobin (HB) and diabetic retinopathy (DR) in patients with type 2 diabetes mellitus (T2DM), and the potential impact of disease duration.</p> Methods <p>Data were collected from the National Health and Nutrition Examination Survey (NHANES) (2001–2018) and the National Metabolic Management Center (MMC) of Fujian Medical University Union Hospital (2018–2023). DR risk factors were identified by univariate analysis. The HB-DR correlation was explored by multivariate logistic regression and analyzed by generalized additive model and smoothed curve fitting. The impact of diabetes mellitus (DM) duration on the correlation was examined by interaction tests.</p> Results <p>Data of 5,089 and 1,893 participants were respectively recruited from the NHANES and MMC databases. In both databases, HB levels were negatively correlated with DR. In the MMC, the negative association persisted in subgroup stratification of sex, age, and body mass index, with the normal weight group reporting a U-shaped relationship at an inflection point of 12.7 (g/dL). The interaction between DM duration and HB significantly impacted the DR probability.</p> Conclusions <p>DM duration may impact the association between HB and DR risk and a low HB level indicates a significant DR risk in patients with T2DM, especially in those with a short DM duration, highlighting the necessity of hemoglobin detection and anemia intervention in the treatment of patients with T2DM.</p> Clinical trial number <p>Not applicable.</p>