We developed two-step thermoresponsive membranes that have two different lower critical solution temperatures. To achieve this, poly(2-dimethylaminoethyl methacrylate)-block-poly(N-isopropylacrylamide) (PDMAEMA-b-PNIPAM, PDN) was introduced into a polyethersulfone (PES) ultrafiltration membrane. The combined Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, attenuated total reflection Fourier-transform infrared spectroscopy, and field-emission scanning electron microscopy results showed that PDN was successfully synthesized, and introduced into the PES membrane. The hydration capacity changes showed that the PES membrane with introduced PDN (PES/PDN) was temperature responsive, and that its pore structure changed. Water flux and protein separation experiments showed that the permeation properties of the PES/PDN membrane effectively changed, and protein separation was achieved based on pore structure changes in response to temperature changes. In particular, PES/PDN containing 5% PDN compared to PES separated water and three types of protein with high efficiency via three clear separation steps, at 30, 50, and 70 degrees C. In addition, the thermoresponsive polymer block in the PES/PDN membrane improved the flux recovery ratio from 29% to 69%.