We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N^N)(CO)3(py-PEG-NH2)](PF6) (py-PEG-NH2 = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MWPEG = 5000 Da, PDIPEG <1.08; N^N = 1,10-phenanthroline (phen) (1-PEG-NH2), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2-PEG-NH2), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3-PEG-NH2)) and [Re(bpy-PEG)(CO) 3(py-NH2)](PF6) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4′-methyl-2, 2′-bipyridine; py-NH2 = 3-aminopyridine) (4-PEG-NH2) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N^N)(CO)3(py-Et-NH2)](PF6) (py-Et-NH2 = 3-amino-5-(N-(ethyl)aminocarbonyl) pyridine; N^N = phen (1-Et-NH2), Me4-phen (2-Et-NH2), Ph2-phen (3-Et-NH2)) and [Re(bpy-Et)(CO)3(py-NH2)](PF6) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4′-methyl-2,2′-bipyridine) (4-Et-NH2). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC50 = 6.6 to 1152 μM) than their PEG-free counterparts (IC50 = 3.6 to 159 μM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH2-4-PEG-NH2 have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N^N)(CO) 3(py-PEG-NCS)](PF6) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-ethoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N^N = phen (1-PEG-NCS), Me4-phen (2-PEG-NCS), Ph2-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)3(py-NCS)] (PF6) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N^N)(CO)3(py-PEG-Bu)](PF6) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl) aminocarbonyl)pyridine; N^N = phen (1-PEG-Bu), Me4-phen (2-PEG-Bu), Ph2-phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO) 3(py-Bu)](PF6) (py-Bu = 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA-4-PEG-BSA and 1-PEG-PEI-4-PEG-PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH2 and its PEG-free counterpart 3-Et-NH2 has been investigated using zebrafish embryos as an animal model. Embryos treated with the PEG complex at high concentrations revealed delayed hatching, which has been ascribed to hypoxia as a result of adhering of the complex to the external surface of the chorion.