A new sandwich-type immunoassay method utilizing high-performance liquid affinity chromatography (HPLAC) and fluorescence-labeled antibodies (FLAs) was proposed and applied to the measurement of concentrations of human serum albumin (HSA) in a buffer and a fermentation broth. In this method antibodies with higher association constants than unfractionated antibody were obtained by fractionation with affinity chromatography and used for fluorescence labeling. It was possible to determine the concentrations of HSA within 30 min, and higher sensitivity and reproducibility were obtained in comparison with the direct method.