The study presents a newly-developed method for extraction-spectrophotometric determination of Co (II) in samples by means of iodnitrotetrazolium chloride (INT). According to our studies, the electronic spectrum of the cobalt ion associate has a peak at 630 nm. The INT:[Co (SCN) 4] ratio in the triple ion-associate complex was 2: 1, and due to the good solubility of the Co (II) ion associate and INT in 1, 2-dichloroethane, maximum extraction into the organic phase was achieved for 30 s. The molar absorptivity of the studied ion associate INT2 [Co (SCN) 4] was ε630= 0.6× 103 L/mol cm. Sandell’s sensitivity of the method was 9.8× 10-2 µg/cm2. The maximum quantity of Co was extracted with INT at pH 2-7. Maximum absorption of the extracts was registered at thiocyanate ions concentration (≥ 1M) in the aqueous phase. The ion associate displayed the highest absorption at INT concentration≥ 6× 10-4 М. The relationship between Co (II) concentration and absorption was linear in the range of 6–125 µg Co (II) in 10 ml aqueous solutions. The content of Co in Amanita calsarea was 1.543±0.158 mg/kg dry weight, in Boletus pinophilus-0.257±0.042 mg/kg dry weight and in Burley tobacco-1.967±0.229 mg/kg dry weight. The extraction-spectrophotometric method for cobalt determination in biological samples with iodnitrotetrazolium chloride, developed in the present study, is characterized with high speed, selectivity and satisfactory accuracy.