Cadmium-free and NIR fluorescent QDs are promising candidates for bio-application. Thus, we present the synthesis of ternary ZnCuInS2/ZnS (ZCIS/ZnS) quantum dots (QDs) where the molar variation of Cu/Zn of the precursors was used to tune the optical and structural properties. QDs with Cu/Zn molar ratio of 2/1 passivated with ZnS exhibited the best optical properties. They showed dominant near-infrared photoluminescence (approx. 850 nm) and highest quantum yield (approx. 52 %, λexc = 500 nm). Therefore, they were further subject to modification to ensure their transfer to the aqueous phase and improve biocompatibility. For this, different functionalization approaches were used. The first method relied on encapsulation with polymers like PSMA (poly(styrene co-maleic anhydride)) and PMAO (poly(maleic anhydride-alt-1-octadecene) coupled with polyetheramine (JEFF; Jeffamine M-1000), and the second relied on hydrophilization with PMAO. Furthermore, we also applied a surface ligand exchange process using DHLA (dihydrolipoic acid) and polyethylene glycol (PEG)-appended DHLA. The comprehensive study indicated that ZnCuInS2/ZnS QDs functionalized with PMAO (ZnCuInS2/ZnS@PMO) exhibited the highest photoluminescence (PL QY) along with ensured high colloidal stability in aqueous media. Moreover, no noticeable deterioration of the photoluminescence profile was observed for all used functionalization approaches. However, a significant decrease in QY was observed for almost all functionalized QDs except those that were PMO-capped. The synthesized QDs were systematically characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD), UV–Vis absorption spectroscopy, and fluorescence spectroscopy. Biological studies indicate that the obtained hydrophilic ZCIS QDs are biocompatible and localized intracellularly inside endosomes.