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Praziquantel (PZQ) is a broad-spectrum antiparasitic drug used in mammals and fish. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of PZQ residues. The hapten PZQ-HS produced by introducing an amino group at a benzene ring synthesized the immunogen and the coating antigen with carrier proteins. Highly sensitive monoclonal antibodies (mAbs) were prepared following mouse immunization and cell fusion. Under optimum conditions, the half maximal inhibitory concentration of the cell line 4E9 was 7.4 ng/mL, and the working range was 2.4–22.8 ng/mL. Immunochromatographic strips (ICS) were developed for the rapid detection of PZQ residues in mackerel. The reliability of the ICS assay was verified with PZQ-negative mackerel and confirmed by HPLC/MS. The cutoff value for ICS was 50 ng/mL PZQ. Our findings revealed that ic-ELISA and ICS can be used for the rapid detection of PZQ in mackerel.