This work was focused on the investigation of the temperature- and pH-responsive polyurethane (PU) membranes and their permeability to amino-acids in response to environmental stimuli. The PU membrane was prepared from a wet phase inversion method and a two-step solution polymerization from polycaprolactone diols (PCL), 4,4'-diphenylmethane diisocyanate (MDI), dimethylol propionic acid (DMPA), etc. The chemical structure, phase state, morphology and surface wettability of the membrane were characterized with Fourier transform infrared (FTIR) spectrometer, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and contact angle tester, respectively. The temperature and pH responses of the membrane were investigated by means of an amino-acid permeate experiment. The L-phenylalanine (L-Phe) was chosen as model amino-acids. The permeation of the L-Phe was measured using a dead-end flow filtration at varied temperatures and pH, and characterized by the permeate flux (J) and rejection coefficient (R). J of the L-Phe across the PU membrane increased with increasing temperature and showed a sharp increase when temperature was raised to the crystalline melting temperature (T m ) of the soft segment of PU, while decreased with increasing pH and having a sharp decrease when pH reached the dissociation constant (pK a ) of DMPA contained in PU macromolecules. While, the R behavior of L-Phe was just opposite from the results of J, which decreased with increasing temperature and increased with increasing pH, also showing the temperature and pH responses. Hopefully, the PU membrane with temperature- and pH-controllable permeability has promising prospects in water treatment, membrane separation, drug delivery system, etc.