Monkeypox (mpox), a zoonotic disease, has rapidly spread globally, prompting the WHO to declare it a public health emergency. The long incubation period, early symptoms resembling respiratory infections, and diagnostic challenges hinder timely epidemic control and accurate clinical diagnosis. The monkeypox virus (MPXV) encodes the A29L protein, which binds to cellular heparan sulfate to facilitate infection and serves as a target for treatment and diagnostics. Thus, developing effective diagnostic tools and treatments is critically important. In this study, we expressed and purified Escherichia coli-derived A29L protein, which was used for chicken immunization to generate specific polyclonal IgY antibodies. The results demonstrated a successful elicitation of a humoral immune response. Subsequently, two single-chain variable fragments (scFv) antibody libraries were constructed using phage display technology, comprising 2.6 × 10