In this work, we have developed a double‐layer nanofiber membrane (NFM) with high selectivity based on molecular imprinting technique by electrospinning methods and applied it in recognizing putrescine. Poly(vinyl alcohol) (PVA) was used as spinning solution, 1,4‐butylene glycol as the imitate template, and ninhydrin as chromogenic agent. The adsorption of the putrescine is corresponded to purple‐color stains on the NFM, which is digitizing intuitively by the ImageJ software. By quantifying the gray value of the stains on the NFM, its imprinting efficiency (IE) is estimated. The adsorption performance of the double‐layer molecular imprinted NFM was studied in detail, showing higher adsorption capacity (71.912 mg mL−1), shorter adsorption equilibrium time of 40 min and higher selectivity to putrescine than our previous works. Furthermore, the material showed remarkable selectivity for putrescine with respect to other three kinds of aliphatic amine in similar molecular structure. The selectivity coefficient (α) of putrescine against cadaverine, spermine, and spermidine are calculated to be 1.734, 2.224, and 2.576, respectively. In addition, since PVA is a water‐soluble material, the NFM is not reusable. © 2020 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48932.