Abstract: A method for the simultaneous analysis of 3 microcystins (MC-RR, MC-YR and MC-LR) was set up by the nano-flow chip liquid chromatography-high resolution mass spectrometry. Under optimal conditions, the mentioned microcystins were baseline separated in 16 min with an ESI source in the positive ion mode. Good linear range in 2.5~2 000 ng/mL was obtained, with the correlation coefficient larger than 0.996 5 and the limit of detection between 0.5~2.0 ng/mL. Solid phase extraction method was used for the extraction and clean-up of fish and shrimp samples. No residue of the three microcystins was detected and the spiked recoveries were between 82.8%~120.3%. The developed method is sensitive, rapid and exact with low sample consumption, so it is reliable for the trace determination of microcystins in biological samples including aquatic products.