Covalent organic framework (COF) film-based solid-phase extraction (F-SPE) has garnered great attention in sample pretreatment. However, harsh synthesis conditions of COF films have severely hindered their potential applications. In this study, a kind of COF (TPB-DMTP) films were fabricated via a liquid-liquid interfacial synthesis method at a mild condition. The obtained films exhibited excellent extraction performance towards microcystins (MCs, an algal toxin) due to their porous structure, high specific surface area and abundant accessible adsorption sites. Coupled TPB-DMTP films-based F-SPE with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), a sensitive and environment-friendly analytical method was established for MCs detection. Under the optimal conditions, this method possessed wide linear ranges (2.0–800.0 pg mL−1) with good linearity (R ≥ 0.9991), low limits of detection (0.8–3.0 pg mL−1) and satisfactory precision (RSDs ≤7.1 %), which then successfully applied for MCs detection in actual aquatic organism samples. Trace amounts of MC-RR (42.4 pg mL−1) and MC-YR (14.6 pg mL−1) were detected in the mussels. The results demonstrate the excellent application potential of COF films in sample pretreatment.