To improve the imprinting efficiency of hyperoside (HYP), the theoretical calculation and UV spectral determination of HYP, acrylamide (AM), itaconic acid (IA), and its different molar ratios of HYP-AM and HYP-IA complexes were carried out by the method of density functional theory (DFT) and the experiment of ultraviolet-visible absorption spectroscopy (UV-Vis). The imprinted active sites of HYP, AM and IA were predicted by using the molecular electrostatic potential (MEP). The imprinting mechanism of the interaction between HYP and AM (IA) molecules was discussed. The results show that O11 of HYP can form strong hydrogen bonds with H9 (or H10) of AM, and H2 (or H15) of IA. While O12—H13, O15—H16, O25—H26, O27—H28, O41—H42, O44—H45, O47—H48 and O52—H53 can form strong hydrogen bonds with O7 of AM, and O4 (or O13) of IA. The high molar ratios of HYP-AM and HYP-IA complexes can increase the total interaction energy of the complex. The strength of the interaction between the active sites can be enhanced by selecting different functional monomers. The strong hydrogen-bond interaction between HYP and AM (IA) leads to partial redshifts of the absorption peak of Ⅰ, Ⅱ and Ⅳ. Moreover, the accuracy of DFT method was verified. The optimal imprinting molar ratios of HYP-AM and HYP-IA complexes were 〔n(HYP):n(AM)=1:8〕 and〔n(HYP):n(IA)=1:7〕, respectively. HYP-IA has stronger interaction with HYP-AM, and IA is a better functional monomer.