As a kind of novel polyelectrolyte materials, poly(ionic liquids) (PILs) have excellent potentiality in the separation of ampholytes suchlike amino acids. Herein, the β-cyclodextrin-based PILs had been synthesized, followed by blending with poly(vinylidene fluoride) (PVDF) to prepare poly(ionic liquid) membranes (PILMs). The Fourier transformed infrared (FT-IR) spectroscopy, 1H nuclear magnetic resonance (1H NMR), scanning electron microscope (SEM), thermal gravimetric analysis (TGA), mechanical strength, zeta potential, and pore size distribution were characterized for these membranes or their intermediates. These PILMs had been developed for the selection separation of the amino acids mixture of L-glutamic acid (L-Glu) and L-threonine (L-Thr). The factors influencing the separation result were investigated, including the solution pH of the amino acids mixture and the PVDF amount in PILMs. The maximum selectivity of 55% can be obtain by one-step separation under the optimal conditions. This separation process using PILMs had the advantages of good selectivity, low operating pressure, larger water flux, and good reusability, which had the potentiality in the separation of various amino acids mixtures.