Abstract: A high sensitive spectrophotometric method for the determination of iodine has been developed. In the pH3.8 KHC₈H₄O₄-HCl buffer solution, in the presence of gum arabic and polyvinyl alcohol,IO₃^- oxidated I^- to produce I₃^-, then I₃^- reacted with methylene blue to form an ion-association. The maximum absorbance is at 525 nm and the molar absorptivity is 1.32×10⁵L/mol·cm, Beer's law is obeyed in the range of 0～30 μg/25 mL. The method has been applied to the determination of iodine in its salt and biological samples with satisfactory results.