Nowadays, extensive research is carried out into finding innovative solutions for the development of stable, reproductible and highly efficient fluorescent contrast agents having the ability of targeting specific cells, which can be further implemented for fluorescent-guided surgery in a real clinical setting. The present study is focused on the development of fluorescent dye-loaded protein nanoparticles (NPs) to overcome the drawbacks of standard administration of free organic fluorophores, such as cytotoxicity, aqueous-instability and rapid photo-degradation. Precisely, Human Serum Albumin (HSA) NPs loaded with two different FDA approved dyes, namely Indocyanine Green (ICG) and Fluorescein isothiocyanate (FITC), with fluorescence response in the near-infrared and visible spectral domains, respectively, have been successfully designed. Even though the diameter of the fluorescent HSA NPs is around 30 nm as proved by Dynamic Light Scattering and Transmission Electron Microscopy (TEM) investigations, they present good loading efficiencies of almost 50% for ICG, and over 30% for FITC and a high particle yield, over 75%. Molecular docking simulations of ICG and FITC within the structure of HSA concluded that the dyes are loaded inside the NPs, docked into Site I (subdomain IIA) of the HSA molecule. After the confirmation of their high fluorescence photostability, the NPs were covalently conjugated with folic acid (HSA-FA NPs) in order to bind specifically to the folate receptor alpha (FRα) protein overexpressed on NIH:OVCAR3 ovarian cancer cells. Finally, fluorescence microscopy imaging investigations validate the improved internalization of folate targeted HSA&FITC NPs compared to cells treated with untargeted ones. Furthermore, TEM examinations of the HSA NPs distribution into the NIH:OVCAR3 cells revealed an increased number of NPs-containing vesicles for the cells treated with HSA-FA NPs, compared to the cells exposed to untargeted HAS NPs, upholding the enhanced cellular uptake through FRα-mediated potocytosis.© 2020 IOP Publishing Ltd.