Background. The kidney is the main excretory organ after diquat absorption. Acute kidney injury (AKI) is a common complication in diquat poisoning patients. Objectives. To identify the value of combined detection of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and neutrophil-lymphocyte ratio (NLR) in the early diagnosis of diquat-induced AKI. Materials and Methods. The retrospective cohort study included 42 diquat poisoning patients. Results. Forty-two patients with diquat poisoning were included, of which 20 had fulminant poisoning (47.6%). At admission (0 h), levels of KIM-1, NGAL, NLR, and acute physiology and chronic health evaluation (APACHE) II scores in the fulminant poisoning group were higher than that of the moderate to severe poisoning group ( P < 0.05), and they were all higher than in the control group ( P < 0.05), while blood urea nitrogen (BUN) and uric acid (UA) levels did not significantly differ across the three groups ( P > 0.05). At 12 h and 24 h, the levels of KIM-1, NGAL, NLR, UA, BUN, and APACHE II scores of patients in the fulminant poisoning group were higher than those in the moderate to severe poisoning group ( P < 0.05), 12 h were higher than 0 h, and 24 h were higher than 12 h ( P < 0.05). Among 42 patients with diquat poisoning, 28 had AKI (66.7%). At 0 h, the AKI group had higher levels of KIM-1, NGAL, NLR, and APACHE II scores than in the non-AKI (NAKI) group ( P < 0.05), while there was no significant difference in BUN and UA levels between the two groups ( P > 0.05). At 12 h and 24 h, the levels of KIM-1, NGAL, NLR, UA, BUN, and APACHE II scores in the AKI group were higher than those in the NAKI group ( P < 0.05), 12 h were higher than 0 h, and 24 h were higher than 12 h ( P < 0.05). KIM-1, NGAL, and NLR are independent risk markers for AKI in diquat poisoning patients. At admission (0 h), the combined application of KIM-1, NGAL, and NLR’s sensitivity, specificity, and area under the curve (AUC) for predicting AKI in diquat poisoning patients was 0.893, 0.859, and 0.903, respectively. Conclusions. KIM-1, NGAL, and NLR can be employed as early diagnostic indicators for the clinical prediction of AKI in diquat poisoning patients. Our findings may help clinicians reduce the occurrence of AKI.