Aims: Urinary haptoglobin (UHp) is a potential biomarker for predicting progress of diabetic kidney disease (DKD) to remedy the defects of currently used urinary albumin. The clinical application of UHp is however limited, owing to the extremely low level in urine. This study aims to establish an enzyme-linked immunosorbent assay (ELISA) kit specifically for detecting UHp in urine samples of patients with diabetes and DKD. Materials and Methods: Supersensitive human haptoglobin antibodies were generated for ELISA kit development, and the sensitivity, specificity and reproducibility of the kit was evaluated. This kit was used to detect UHp in 246 healthy individuals and 83 patients with type 2 diabetes (T2D). The interference of blood haptoglobin genotypes on UHp measurement was analysed. Results: The UHp ELISA kit had a standard curve ranging from 5 to 200 ng/ml. The low detection limit was 0.11 ng/ml. The coefficients of variation of intra- and interassay were 5.5% and 8.3%, respectively. The kit showed high accuracy with 100.9% mean recovery rate, and linearity R2 = 0.999. The reference range of UHp was 0–42.3 ng/g creatinine (0–Q95) in the healthy individuals. UHp level was significantly higher in T2D patients with microalbuminuria and macroalbuminuria than that in T2D without microalbuminuria (p < 0.01). The UHp concentration measured by this kit was not affected by haptoglobin genotypes. Conclusions: We have generated an ELISA kit to accurately detect UHp levels, which is potentially a reliable biomarker of DKD.