Fast and sensitive detection of microcystin-LR (MC-LR) was conducted with a portable trace organic pollutant analyzer (TOPA) based on the principle of immunoassay and total internal reflection fluorescence. The reusable fiber optic probe surface was produced by covalently immobilizing a MC-LR-ovalbumin (MC-LR-OVA) conjugate onto a self-assembled thiol-silane monolayer of fiber optic probe through a heterobifunctional reagent. It has been established that the MC-LR-OVA immobilized fiber optic probe is highly resistive to non-specific binding of proteins, while bound anti-MC-LR antibody can easily be eluted from the immunosurface with high recoveries (more than 150 assay cycles) in the way of pepsin solution, without any damage to the surface-immobilized MC-LR derivatives. One assay cycle including surface regeneration was less than 20 min. For the standard curve, the limit of detection (LOD) of 0.03 microg/L and the quantitative detection range of 0.1-10.1 microg/L was obtained when the concentration of antibody labeled by Cy5.5 was 0.28 microg/mL. Cross-reactivity against a few compounds structurally similar to MC-LR was little. The developed immunosensor method was applied to the monitoring of MC-LR in various types of water. The recovery of MC-LR added to water samples at different concentrations ranged from 80 to 110% with relative standard deviation (R.S.D.) values less than 5%. The immunoassay performance of the TOPA was validated with respect to conventional high-performance liquid chromatography, and the correlation between methods was in good agreement (r(2)=0.9978). The TOPA is a portable, easy-to-use, and robust immunoassay system and commercially obtained from the company JQ-environ Co. Ltd. (China).