An analytical method for screening aptamers for different recognition sites in lactoferrin (Lac) molecules has been developed based on Surface Plasmon Resonance imaging (SPRi), combined with the cluster classification calculation of a quasi-aptamer library strategy and molecular docking simulation analysis. Using the software simulation, a homology analysis was performed on the selected quasi-aptamer sequences, which could be divided into 8 different families. Based on the principle of biomolecular recognition, a label-free, high-throughput dual immune site screening method was established, in which the nucleic acid aptamers of recognizing ability for lactoferrin molecules were fixed onto the surface of the SPRi sensor chip and could bind to the lactoferrin molecules. Then, the aptamer candidates to be paired were introduced, and the recognition event of the second immune site was judged by observing the binding signal of SPRi. The paired SPRi signal was generated only when the site identified by the second nucleic acid molecule was different from the first immune site. Based on this principle, a pair of Lac nucleic acid aptamers (Lac-8 and Lac-25) was finally screened and confirmed using computerized simulation, and has been employed to assay Lac in milk by ELONA (Enzyme-Linked Oligonucleotide Assay).