Abstract: GeXIVA1,2 is a new type of conotoxin recently discovered in the transcriptome of Conus generalis and it is expected to be used clinically as a new type of analgesic. This study established and verified a sandwich enzyme-linked immunosorbent assay method for the marine drug GeXIVA1,2 in the plasma of rats and Beagle dogs. The mouse monoclonal antibody 4B2 and biotin-labeled rabbit polyclonal antibody 2# were developed. The checkerboard method was used to optimize the antibody pairing concentration, minimum dilution ratio, incubation temperature, and incubation time to establish an antibody sandwich ELISA detection method. Verify the established testing methods. The established ELISA method has a quantitative range of 1.25-80 ng�mL^-1 in rat and Beagle plasma. The precision, accuracy, selectivity, specificity, stability, dilution linearity, and hook effect all meet the requirements for biological sample analysis. All the procedures for the animal experiments were approved by the Animal Ethics Committee of the Institute (Permit Number:IACUC-DWZX-2020-698). This method can support the preclinical pharmacokinetic study of the marine drug GeXIVA.