This study explores the synthesis and characterization of aggregation-induced emission enhancement (AIEE)-active gold nanoclusters (AuNCs), focusing on their near-infrared luminescence properties and potential applications in biological imaging. These AIEE-active AuNCs were synthesized via the NaBH4-mediated reduction of HAuCl4 in the presence of peptides. We systematically investigated the influence of the peptide sequence on the optical features of the AuNCs, highlighting the role of glutamic acid in enhancing their quantum yield (QY). Among the synthesized peptide-stabilized AuNCs, EECEE-stabilized AuNCs exhibited the maximum QY and a pronounced AIEE effect at pH 5.0, making them suitable for the luminescence imaging of intracellular lysosomes. The AIEE characteristic of the EECEE-stabilized AuNCs was demonstrated through examinations using transmission electron microscopy, dynamic light scattering, zeta potential analysis, and single-particle imaging. The formation of the EECEE-stabilized AuNCs was confirmed by size-exclusion chromatography and mass spectrometry. Spectroscopic and electrochemical examinations uncover the formation process of EECEE-stabilized AuNCs, comprising EECEE-mediated reduction, NaBH4-induced nucleation, complex aggregation, and subsequent cluster growth. Furthermore, we demonstrated the utility of these AuNCs as luminescent probes for intracellular lysosomal imaging, leveraging their pH-responsive AIEE behavior. Additionally, cyclic arginylglycylaspartic acid (RGD)-modified AIEE dots, derived from cyclic RGD-linked peptide-induced aggregation of EECEE-stabilized AuNCs, were developed for single- and two-photon luminescence imaging of αvβ3 integrin receptor-positive cancer cells.