This study focuses on enhancing the sensitivity of lateral-flow strips (LFSs) based on gold nanoparticles (AuNPs) for the detection of Neutrophil Gelatinase-Associated Lipocalin (NGAL) protein in urine samples. Several sizes of AuNP-based LFS biosensors were tested to optimize colorimetric signals for NGAL detection based on improved conjugation conditions. AuNPs of 39.8 nm diameter at pH 8 were the most sensitive for the detection of NGAL. Through systematic enhancements to the AuNP-based LFS, the study significantly improves the sensitivity, enabling the reliable detection of NGAL protein in urine samples at a level as low as 12.5 ng mL-1. These advances contribute to the refinement of diagnostic tools for the early detection of kidney injury, specifically in cases associated with the presence of NGAL protein, offering a more precise and effective screening approach.