Developing a system that combines active targeting to specific cells, enhanced cellular uptake, photothermal transduction capacity, and biocompatibility is a challenge to make nanoparticles applicable in the field of biomedicine. In this study, we carried out the functionalization of gold nanoparticles (AuNP) in several steps strategically using macromolecules to provide key characteristics of theragnostic agents. Polyethylene glycol (PEG), being hydrophilic, enhances nanoparticle stability and circulation lifetime. Polylactic acid (PLA), which is a biodegradable hydrophobic polymer, plays an important role in the interaction and uptake of these nanoparticles through cellular membranes. Furthermore, functionalization with folate can offer active targeting, as tumor cells typically overexpress folate receptor proteins. By single, double, and triple functionalization of spherical and rod-shaped AuNP with these ligands, we obtained varying properties related to aggregation, stability, and localized surface plasmon resonance (LSPR). Triple functionalization ensured simultaneous stability of the nanoparticles in aqueous media and a significant increase in cellular uptake. Additionally, the incidence of infrared radiation reveals that nanorods can increase the temperature more effectively gold nanospheres due to their longitudinal surface plasmon resonance band. The results suggest that this functionalization strategy can be employed to fine-tune desired properties, enabling practical and effective applications of gold nanoparticles in imaging and photothermal therapy within biomedical research.