BACKGROUND: Recent advances in nanotechnology have led to the use of nanomaterials in the diagnosis of cancer by imaging techniques. OBJECTIVE: This study aimed to synthesize fluorescein-conjugated gold nanoparticles and study the parameters affecting the loading of fluorescein on synthesized coated gold nanoparticles with the ability to be used in medical diagnostic methods. METHODS: The synthesized gold nanoparticles were functionalized with polyethylene glycol. Then, these particles were conjugated with fluorescein under different conditions. To investigate the optical and structural features as well as the factors affecting the loading, the nanoparticles were evaluated by ultraviolet-visible, fluorescence and FT-IR spectrophotometer, fluorescence spectrophotometer, transmission electron microscopy, dynamic light scattering, and zeta potential measuring device. Also, the use of these particles in cancer diagnosis on the skin melanoma cell (B16F10) was examined using a fluorescence microscope. RESULTS: PEG-coated spherical gold nanoparticles were synthesized as a carrier for the fluorescein dye detector. The coating agent concentration, incubation time, temperature, and pH of the medium affected the loading efficiency of fluorescein on the nanoparticles. Also, optimal conditions for use in the diagnostic applications were investigated. Ten micromolar of the sample were selected for cell imaging studies. The fluorescence signal of B16F10 cells containing nanoparticles was relatively strong, indicating the amount of nanoparticles uptaken by the cells. CONCLUSION: The results showed that by designing fluorescent gold nanoparticles with fluorescein as fluorescent detectors and considering their diagnostic importance, an efficient way to diagnose incurable diseases can be found.