Biocompatible L-glutamic acid stabilized gold nanoparticles (Glu-AuNPs) were synthesized, characterized, and applied in a pH cooperative improved colorimetric detection of Cr(III) ions. The as-prepared Glu-AuNPs were characterized by TEM, DLS, XRD, and zeta potential analysis and MTT assay with IC50 investigations to confirm its low-toxicity. In contrast to the case of without any pH buffer, the Glu-AuNPs revealed enhanced colorimetric response to Cr(III) ions from 400 nM onwards at pH 6. In the presence of Cr(III) and at pH 6, the Glu-AuNPs displayed a new absorbance peak at 641 nm accompanied with the diminishing of its origin peak at 525 nm. The enriched particles aggregation and binding forces behind the pH cooperative tactic were proposed through the TEM, DLS, zeta potential, and FTIR interrogations. At pH 6, the Glu-AuNPs delivered a linear response from 0.01 to 2 µM for Cr(III) with a LOD of 0.76 ± 0.013 nM. This pH supportive strategy was further supported by the tap and lake water samples investigations, which showed exceptional recoveries (105–112% and 104–115%, respectively) with values comparable to the ICP-mass inquiry.