The present study elucidates the morphology characteristic of nylon‐6 as protein immobilization film and their influences in biological recognition interface for a single‐use electrochemical immunosensor. A basic requirement of this polymer platform is to allow the testing analyte [conductive polyaniline‐Fe2O3‐glutaraldehyde‐conjugated‐N‐hydroxysuccinimidobiotin (PANI‐Fe2O3‐GA‐b‐NHS)] to access to the immobilized capture reagent [bovine serum albumin (BSA)] on a surface of the nylon‐6 film. As an immunoassay, the biochemical reaction between PANI‐Fe2O3‐GA‐b‐NHS and BSA takes place on a surface of the nylon‐6 support film, which then translated into measurable resistance signal through pulse‐mode measurement. The in‐house developed nylon‐6 film, N‐16B, with 16 wt % nylon polymer and methanol as nonsolvent had demonstrated the fastest lateral wicking speed (1.07 mm s−1) and excellent protein immobilization capacity (1650 ± 85.84 μg cm−3), leading to the higher electrical current signal with a minimal resistance of electrochemical responses (0.57 ± 0.04 MΩ). This work reflects the importance of the physical characteristics of the nylon‐6 film that determines the sensitivity and effectiveness of an immunosensor. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46741.