Neutrophil gelatinase-associated lipocalin (NGAL) is an important biomarker for the early diagnosis of acute kidney injury (AKI). Herein, a sensitive dual-signal sandwich-type electrochemical immunosensor was designed for NGAL detection using square wave voltammetry (SWV) and current-time (i-t) curves. Herein, MXene-loaded polyaniline (MXene/PANI) nanocomposites were fabricated and utilized as the sensing platform for anchoring gold nanoparticles (Au NPs) and immobilizing primary antibodies. Subsequently, an Au NP-loaded copper metal-organic framework decorated with a single-walled carbon nanohorn (Au/Cu-MOF/SWNH) nanocomposite was prepared by depositing Au NPs onto Cu-MOF/SWNHs. The NGAL affinity peptide (Pep) was bound to Au/Cu-MOF/SWNHs to form a Pep/Au/Cu-MOF/SWNH nanocomposite as a probe. The probe exhibited an SWV signal (signal 1) wherein electron transfer between Cu2+ and Cu+ of the Cu-MOF without the extra electron transport medium was observed. The i-t signal (signal 2) was obtained from the reduction of H2O2 by the Au/Cu-MOF/SWNH nanocomposite with high electrocatalytic activity. Au/Cu-MOF/SWNH nanomaterials exhibit optimal analytical performance in the linear range of 0.00001-10 ng-mL-1 with limits of detection of 0.0074 pg-mL-1 (SWV) and 0.0405 pg-mL-1 (i-t) for NGAL determination. The immunosensor was further used to detect NGAL in the sample, which has potential applications in AKI diagnosis.