Fiber separation technology has been widely used in the removal of metal ions, dyes, proteins, and particles. In this work, the reversible characteristic of temperature sensitive hydrogel which possesses the expansion and contraction at different temperature is used to control the change of internal pores of filter materials for achieving the selective separation and concentration of solution. We prepared polylactic acid (PLA) hollow fiber as outer support layer by the template method and PLA electrospun membrane as internal support diaphragm by electrospinning. Then, the method of adding poly(N‐isopropylacrylamide) (PNIPAM) hydrogel in the hollow fiber layer by layer was adopted to form the hydrogel/PLA fiber. When the mass ratio of PNIPAM/PLA was 3/1, the sample presented the relatively tight interior morphology and was selected for protein separation. By adjusting the temperature, the rejection capacities of PNIPAM/PLA composite fiber for bovine serum albumin (BSA), and ovalbumin (OVA) were in the range of 25–36 mg/g and 22–31 mg/g, respectively. Furthermore, the fiber reusability was investigated by swelling and deswelling elution processes. It was found that the sample could be reused 5 times. This work explores a novel control method for the graded filtration and concentration of targets.