Microcystins are a group of cyanotoxins which interact with the C-terminal region of PP1 and PP2A proteins, so denaturation and inactivation are necessary for breaking covalent binding to release microcystins. In this study, a novel extraction method was developed by laser irradiation desorption of microcystins from fish protein. The sample was mixed with aqueous methanol and irradiated by a 450 nm laser, with an optimized value of laser power density at 8 W and exposure time at 5 min. ThenLC-MS/MS was applied for the determination of microcystins in fish extracts. The ionization behaviors of microcystins were investigated firstly, and doubly charged microcystins were selected as precursor ions in multiple reaction monitoring scan for quantification. This proposed quantitative method was well validated in terms of selectivity, linearity, sensitivity, accuracy, recovery, and stability. The successful application of this LC-MS/MS method showed its ability for the analysis of microcystins in low concentration, and it would be of significant interest for environmental and food safety applications to ensure the safety of fish and related products.