Fluorescein-loaded magnetic nanoparticles (FMNPs) have been increasingly utilized in nanomedicine due to their unique properties. In this study, polyamidoamine (PAMAM) dendrimer was used to modify the FMNPs through bifunctional polyethylene glycol linker. The obtained PAMAM modified magnetic nanoparticles (PFMNPs) were characterized by transmission electron microscope, thermogravimetric analysis, zeta potential titration, and fourier transform infrared spectroscopy. The effect of PAMAM conjugation on the biodistribution of FMNPs and PFMNPs were investigated by confocal laser scanning microscopy and inductively coupled plasma atomic emission spectrometry, respectively. It was revealed that PAMAM conjugation resulted in a lower uptake of FMNPs in the lung and less aggregation in the liver, whereas a higher uptake in brain and testis. Furthermore, the serum biochemical and the hematological analysis indicated the PFMNPs caused no significant changes in enzymes reflective of inflammatory response or organ toxicity.