Ingestion of water contaminated with the cyanotoxin, microcystin (MC), can pose serious health risks to humans. MC is also known to accumulate in seafood; however, this exposure pathway is much less understood. A fundamental element of this uncertainty is related to analytical difficulties. Commercially available enzyme-linked immunosorbent assays (ELISAs) offer one of the best options for routine MC detection, but methods of detecting MC in tissue are far from standardized. We spiked freshwater finfish and marine mussel tissues with MC, then compared recovery rates using four different preparation protocols and two ELISA types (polyclonal anti-MC-ADDA/direct monoclonal (DM)). Preparation protocol, type of ELISA, and seafood tissue variety significantly affected MC detection. This is the first known study to use DM ELISA for tissue analyses, and our findings demonstrate that DM ELISA combined with a short solvent extraction results in fewer false positives than other commonly used methods. This method can be used for rapid and reliable MC detection in seafood.