Carbon dots (CDs) are promising for agro-environmental applications; however, clear connections between synthesis, photophysical properties, size, and biosafety are often not well established. In this study, we map these relationships for glucose–arginine CDs (GA-CDs). By using microwave and hydrothermal routes at precursor ratios of 1:3, 1:9, and 1:15, we produced sub-10 nm nanoparticles (analyzed by dynamic light scattering and atomic force microscopy) that exhibit tunable absorption and emission properties, as well as surface properties (demonstrated through UV–Vis spectroscopy, 3D photoluminescence, and FTIR analysis). The hydrothermal 1:9 condition yielded the narrowest size distribution and red-shifted photoluminescence. Across biological models spanning plants, insects, plant-growth-promoting bacteria (PGPR), and human cells, GA-CDs were well tolerated, with no adverse changes detected in plant stress markers, aphid feeding behavior or fecundity, or PGPR growth. In A549 cells, viability remained stable up to a concentration of 0.125 mg mL⁻¹, while exposure to 0.5 mg mL⁻¹ reduced viability, establishing a practical operating range. These results provide a clearer picture of how the structure and properties of carbon dots derived from arginine and glucose are correlated to their safety. The GA-CDs are, therefore, useful, and traceable tools for agro-environmental research. The findings support their use as biocompatible nanomaterials for studying interactions among plants, insects, and microbes in agriculture.