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Aim: To develop probes for detecting the binding specificity between β-secretase and substrate, and provide reliable biological activity data for further researching encircling substrate-based inhibitors. Methods: To prepare the inhibitors, the hydroxyethylene (HE) segment including P1 and P1’ was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human β-secretase ectodomain (amino acid residues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this αβ-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and molecular mechanics-generalized-b surface area (MM-GBSA) analysis, were performed on the complex of β-secretase and OM99-2 to obtain the geometrical and energetical information. Results: We designed and constructed a positional scanning combinatorial library including 16 compounds; all members of the library were synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P4-P1 and P1’-P4’ positions led to the discoveries of P and P’ sides binding specificity and potent inhibitors 14, 18, and 22. The binding free energy on the whole system and every residue were compared to the biological assay result. Conclusion: The removal of P4’ yielded inhibitor 22 (A3*B2) with high potency; further truncation of P3’ gave inhibitor 18 (A3*B1) with equal activity, implying that the right side of the inhibitors play a less important role and could be easily simplified, while change on the P side may cause substantial results.