In this study, we first reported a lateral flow assay combined with primer extension (PEXT) and gold nanoparticles for single-nucleotide polymorphism (SNP) genotyping of the tmigd1 gene of the Tsaiya ducks (Anas platyrhynchos), which has the advantages of simplicity of operation, cost-effectiveness, and time-saving. Gold nanoparticles were tailed with thiol-thymine oligodeoxyribonucleotides (thiol-(dT)30) using the salt-aging method at 25°C and used as a label in a lateral flow assay. The lateral flow device was composed of test and control zones on a nitrocellulose membrane containing streptavidin and adenosine oligodeoxyribonucleotides ((dA)30), respectively. When the specific SNP existed, the corresponding primers were extended, and the reaction product was captured by streptavidin at the test zone owing to the introduction of biotin-deoxyuridine triphosphate (biotin-dUTP) into the reaction product during PEXT. Gold nanoparticles hybridized with the reaction product to render it visible. Here, we developed a new system for detection of single nucleotide polymorphism in a female reproduction-associated gene, tmigd1, of Anas platyrhynchos using the strip biosensor, and identified the optimized parameters for the concentration of Mg2+ in the PEXT reaction and the amount of streptavidin used on membranes for signal specificity.