17β-Estradiol (E2) is one of the most significant growth-promoting steroid hormones that can be used illegally in livestock, and strict monitoring of this hormone is critical for the safety of meat consumers. A rapid immunochromatographic lateral flow test strip was developed in a competitive format using the gold-conjugated polyclonal antibody (pAb) to specifically and rapidly determine the residues of E2 as a steroid growth promoter hormone in sheep serum. The test procedure was carried out within 5 min, and the results of this qualitative one-step assay were examined visually according to whether test lines appeared or not. The cutoff limit of the strip test was determined at 1 ng/mL by the naked eye. Analysis results of E2 pAb showed no cross-reactivity with other steroid hormones. Therefore, the described lateral flow test strip can be used as an instrument-free, rapid, cost-effective and on-site screening tool for the simultaneous determination of E2 in sheep serum.