A new method for the determination of cobalt was established by spectrophotometry with 5’-nitro-salicylfluorone(5’-N-SAF)as coloring agent. In the buffer solution of NH₃.H₂O-NH₄Cl at pH 9.2, cobalt(Ⅱ) reacted with 5’-N-SAF and formed a red complex in the presence of TritonX-100. The apparent molar absorptivity was 3.7 *10⁴ L.mol^-1.cm^-1 at 546 nm; the detection limit(3s/k)was 5.64 *10^-3 mg/L; Beer's law was obeyed for the mass concentration of cobalt(Ⅱ)in range of 0~0.5 μg/mL. The method was applied to the determination of cobalt content in Vitamin B₁₂ injections, and the results were satisfactory.