DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of promising fluorophores for imaging and sensing applications. All aspects of their spectroscopic properties are not yet fully characterized, leaving this field still with a number of fundamental studies to be addressed. In this work, we studied the spectroscopic properties of red-emitting DNA-AgNCs at different pH (5 to 9) and ionic strength μ (0.005 to 0.5). The photophysical properties of high performance liquid chromatography (HPLC) purified DNA-AgNCs proved to be constant over a large range of pH and μ, with absorption, emission and fluorescence decay times only being affected at very high pH and μ values. Non-purified DNA-AgNCs were also unaffected by pH and/or μ variations, but significant differences can be observed between the rotational correlation times of purified and non-purified DNA-AgNCs.