Trichinella spiralis (T. spiralis) is a zoonotic parasite that is seriously harmful to livestock and humans, causing large number of infection cases annually. Currently, there is an urgent need for sensitive and specific assays for T. spiralis antibody testing to control zoonotic spread from animals to humans. Herein, gold nanoparticle (AuNP) probes were dual-functionalized with oligonucleotides and anti-cystatin-like protein (CLP) monoclonal antibodies (Mabs). AuNP probes competed for CLP with target serum antibodies to form immune complexes, which were captured by polyclonal antibody-modified magnetic beads followed by convenient magnetic separation. The hybridization chain reaction (HCR), a form of enzyme-free nucleotide amplification at room temperature, was triggered by AuNP probes to amplify the signal output. The ultrasensitive competitive immunosensor enhanced by HCR (Au@Mab-I1-iHCR) exhibited a low detection limit of 31.7 ng mL−1, which was 5.5-fold lower than that of a commercial enzyme-linked immunosorbent assay kit, and no cross-reaction in other parasite- or virus-infected serum. The proposed immunosensor showed 97 % sensitivity and 99 % specificity in human serum and good diagnostic performance in artificially infected swine serum. In light of its time-efficient process (125 min), sensitivity and selectivity, the Au@Mab-I1-iHCR has potential applications for T. spiralis antibody monitoring in herds and humans for zoonosis screening.