On the basis of affinity of Fe(III) ions toward functional groups of 2,6-dihydroxy benzoic acid (2,6-DHB), a charge transfer complex (CT) is formed between Fe(III) ion and 2,6-DHB in acidic media. The stoichiometry and stability constant of the complex was determined using both mole ratio and Job’s methods. The stability constant of 1:1 complex formed in acidic media is 2.55±0.14×105(mole ratio) or 2.70±0.12×105(Job), in which the quantities are in agreement with together. This method was used for spectrophotometric determination of Fe(III) in aqueous media. The dynamic range and detection limit (DL) were 1.2×10-5to 3.6×10-4M and 1.5×10-6M, respectively. The relative standard deviation (RSD) was found to be 0.76% in the replicate treatments (n=7) with 1.6×10-4M Fe(III). The practical applicability of the method was confirmed using a synthetic certificated reference material (CRM) and spiked natural water samples.