Neutrophil gelatinase-associated lipocalin (NGAL) is a candidate diagnostic biomarker for acute kidney injury (AKI). Since there is no specific treatment to reverse AKI, a good biomarker such as NGAL can increase the performance of clinical care. Therefore, a timely, specific and sensitive assay for detecting NGAL is critical for clinical determination. In this study, we established a solid-phase proximity ligation assay for the detection of NGAL using polyclonal antibodies conjugated with a pair of oligonucleotides. The data are read out as the Ct value via quantitative real-time polymerase chain reaction (qPCR). Our results demonstrate that this new assay performs with good specificity and sensitivity for detection of NGAL spiked in buffer or serum, which indicates that the solid-phase proximity ligation technique is a promising tool for diagnostics in clinical decisions.