A simple and convenient preparation method has been developed for polymeric crown ethers containing the dibenzo-18-crown-6 subunit and electrospun composite nanofibers composed of polymeric crown ethers with polystyrene (PCE-PS). Furthermore, this composite nanofiber was used as a sorbent for selective extraction of plasma catecholamines, viz. dopamine (DA), norepinephrine (NE), and epinephrine (E). After the protein deposition of the plasma sample, the supernatant was adjusted to neutral pH with buffer solution. Then, the mixture was loaded and pushed through a column packed with composite nanofiber sorbent, and the analytes were eluted with 50 mu L of acetic acid. The effectiveness of the plasma sample cleanup method was verified by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The PCE-PS packed column may prove clinically useful, as it provides a convenient and selective method for evaluation of human plasma catecholamines.