Phycotoxins produced by several algae, microalgae, and cyanobacteria, are highly toxic contaminants of environmental water and sea foods. They cause necessity of efficient techniques for their simple and rapid detection. In the study, fluorescence polarization immunoassay (FPIA) was developed for microcystin-LR (MC-LR), one of the most widespread phycotoxins. Several fluorescent derivatives of MC-LR were synthesized and compared for this purpose. The reached detection limit for MC-LR was 7.5 ng/mL, and the working range of determined concentrations was 50–500 ng/mL. The developed FPIA provides rapid (10–30 min, including sample preparation) and sensitive testing. The developed portable detectors of fluorescence polarization allow testing environmental objects directly at the sampling points and making decisions about their contamination and safety rapidly.