Interferon gamma (IFN-gamma) has various immunoregulatory effects and its measurement is widely used for monitoring immune response in many clinical trials for cancer vaccines. In this study, we developed a fluorescent-linked immunosorbent assay (FLISA) for the measurement of IFN-gamma. The FLISA can accurately measure IFN-gamma concentrations and the linear dynamic range of standard curve is 15.6-2000 pg/ml (2.1 log range). Because the FLISA needs only a small amount of antibodies and there is no washing process, it is able to reduce hands-on time and running costs compared with traditional enzyme-linked immunosorbent assay (ELISA). The FLISA can be readily performed in both 96- and 384-well plates, and up to 60 plates can be handled automatically by a robotic arm. Measurement of IFN-gamma by fluorometric microvolume assay technology (FMAT) may be a high-throughput screening method for a large number of samples.