Abstract: A rapid and sensitive method has been developed for the determination of Cobalt(II) based on complexation reaction between the metal ion and 3,4,-dihydroxybenzaldehyde-1-(3-Chloro-2-Quinoxalinyl)Hydrazone (DHBCQH) in the presence of non-ionic surfactant Tween-80. The important parameters affecting the analytical procedure were optimized. Absorption maximum for a ternary complex was noted at 450 nm. The reaction was found to be rapid at room temperature and absorbance remained constant for more than 24hs. The method obeys Beer’s law in the range 29.46 to 132.59ng/ml. The apparent molar absorptivity of 4.45×105 L mol-1 cm-1 and Sandell’s sensitivity 0.13ng/ml. The effect of foreign ions was tested by taking a constant concentration of metal ion and determining its concentration in the presence of ≥ 100 folds in excess of foreign ions. The method was successfully used in the determination of Cobalt(II) in Biological samples. Second order derivative spectrophotometric method were developed at λmax = 482nm for the determination of Cobalt(II), which was more sensitive than the zero order method.