Increasing global warming and eutrophication have led to frequent outbreaks of cyanobacteria blooms in freshwater. Cyanobacteria blooms cause the death of aquatic and terrestrial organisms and have attracted considerable attention since the 19th century. Microcystin-LR (MC-LR) is one of the most typical cyanobacterial toxins. Therefore, the fast, sensitive, and accurate determination of MC-LR plays an important role in the health of humans and animals. Immunoassay refers to a method that uses the principle of immunology to determine the content of the tested substance in a sample using the tested substance as an antigen or antibody. In analytical applications, the immunoassay technology could use the specific recognition of antibodies for MC-LR detection. In this review, we firstly highlight the immunoassay detection of MC-LR over the past two decades, including classical enzyme-link immunosorbent assay (ELISA), modern immunoassay with optical signal, and modern immunoassay with electrical signal. Among these detection methods, the water environment was used as the main detection system. The advantages and disadvantages of the different detection methods were compared and analyzed, and the principles and applications of immunoassays in water samples were elaborated. Furthermore, the current challenges and developmental trends in immunoassay were systematically introduced to enhance MC-LR detection performance, and some critical points were given to deal with current challenges. This review provides novel insight into MC-LR detection based on immunoassay method.