Incorporation of highly selective and stable adsorbent with facile extraction technology is desired in practical analysis. Here we show the rational preparation of a urea-linked covalent organic framework functionalized polytetrafluoroethylene film (COF-117-PTFE) with ordered porous structure, rich functional groups, and large surface area-to-volume ratio as the effective adsorbent for convenient, selective and rapid thin film microextraction (TFME) of rhodamine B (RB). The COF-117-PTFE based TFME coupled with high performance liquid chromatography-fluorescence detector (HPLC-FLD) successfully realized the determination of RB with the limit of detection of 0.007 μg L−1, the linear range of 0.1 - 100 μg L−1. The relative standard deviation (RSD) of intraday (n = 5) and interday (n = 5) for the determination of 10 μg L−1 RB were 2.3% and 6.8%, respectively. The absolute recoveries were 80.3%, 71.2% and 67.9% in river water, chili powder and Sichuan pepper powder, respectively. The recoveries for RB spiking in complicated real samples (dry chili, chili powder, dry Sichuan pepper, Sichuan pepper powder and river water) ranged from 90.4% to 107.5%. The developed COF-117-PTFE based TFME-HPLC-FLD method is promising in practical application. This work reveals the high potential of functionalized COF film as the adsorbent for effective extraction of trace contaminants in complicated samples.