In this study, we reported on the synthesis and biological evaluation of radiolabeled fluorescent dye conjugated bovine serum albumin nanoparticles within the size range 190-210 nm. The bovine serum albumin nanoparticles (BSANPs) were prepared using a desolvation method, and chemical cross-linking was performed using gluteraldehyde. Furthermore, pheophorbide-a (PH-A) was loaded on the BSANPs. The results obtained from dynamic light scattering and electron microscopy have proved that nanoparticles are highly monodisperse and near-spherical shaped. The photo-physical properties of the PH-A-BSANPs were obtained using the spectrophotometric techniques. According to the results, PH-A and BSANPs show high non-covalent interaction. PH-A loaded nanoparticles were labeled with Tc-99m and the radio-labeling efficiency was determined as 90 +/- 1.2%. Biodistribution studies of Tc-99m labeled PH-A-BSANPs and PH-A were carried out using female Albino Wistar rats, and Tc-99m-PH-A-BSANPs showed a significantly higher uptake in the breast and uterus than Tc-99m-PH-A. Cell culture study was carried out in MCF-7 cell line ( human breast adenocarcinoma cell line). According to the cell culture studies, Tc-99m-PH-A-BSANPs showed a higher uptake than Tc-99m-PH-A. Moreover, PH-A-BSANPs demonstrated good photo-physical properties and BSANPs increased the uptake of PH-A on to the MCF-7 cell line. These results confirm that Tc-99m labeled PH-A-BSANPs could be utilized for radioimaging. (C) 2011 Elsevier B. V. All rights reserved.