A sensitive and selective spectrophotometric determination of human serum albumin(HSA) was proposed by using the ternary complex-formation reaction among 2-(5-bromo-2-pyridylazo)-5-(N-phenyl-N-sulfopropylamino)phenol (5-Br.PAPS) as a pyridylazo derivative, cobalt(II) and HSA in the presence of poly(N-vinylpyrrolidone) (PVA) as a dispersion agent. The calibration curve was linear in the range of 0 - 7.0 μg/ml HSA by measuring the difference of absorbances at 636 nm between ((5-Br.PAPS)-cobalt(II)-HSA) and (5-Br.PAPS-cobalt(II)) solutions. The sensitivity was > 6-fold over the (Pyrogallol Red(PR)-molybdenum(VI)) method, and the recovery test in urine was satisfactory (97.5 ± 2.8%).