Microcystin‐LR (MC‐LR), as a hepatotoxin, can cause liver swelling, hepatitis, and even liver cancer. In this study, MC‐LR aptamer (Apt‐3) modified graphene oxide (GO) was designed to enrich MC‐LR in white jade snail (Achatina fulica) and pond water, followed by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) analysis. Results indicated that the Apt‐3/PEG/GO nanocomposites were highly specific to MC‐LR, and the detection limit of MALDI‐MS was 0.50 ng/mL. Moreover, the MC‐LR can be released from nanocomposites at 75°C, thus, the reuse of Apt‐3/PEG/GO is realized. Real sample analysis indicated that the Apt‐3/PEG/GO nanocomposites coupled with MALDI‐MS were efficient in detecting trace amounts of MC‐LR in real samples. With the merits of being low cost, reusable, and easy to besynthesized, this Apt‐3/PEG/GO MALDI‐MS is expected to be comprehensively applied by anchoring suitable aptamers for different targets.