A versatile and highly sensitive strategy for nanopore detection of microcystin-LR (MC-LR) is proposed herein based on the aptamer and host-guest interactions by employing a gold nanoparticle (AuNP) probe. The aptamer of MC-LR and its complementary DNA (cDNA) are respectively immobilized on AuNPs with distinct sizes (5 nm AuNPs for the aptamer and 20 nm for the cDNA), and the constructed polymeric AuNP network via the hybridization of the aptamer and cDNA was disintegrated upon the addition of MC-LR The specific interactions between the aptamer and MC-LR disrupt and release the cDNA-AuNPs that were then removed by centrifugation, leaving the MC-LR-aptamer-AuNP species in the supernatant for subsequent nanopore determination. By monitoring the current blockade of released MC-LR-aptamer-AuNPs using a specific tailored nanopore (10 and 20 nm in diameter, generated by current dielectric breakdown), we could deduce the presence of MC-LR, as the bulky NP network could not pass through a nanopore with a relatively smaller size. We realized the detection of MC-LR with a concentration as low as 0.1 nM; additionally, we have proved the specificity of the interaction between the aptamer and MC-LR by replacing MC-LR with other congener toxins (MC-RR and MC-YR), chlorophyll (a component abundantly coexists in water), and the mixture of the four.