Here,　an　imine-linked-based　spherical　covalent　organic　framework　(COF)　was　prepared　at　room　temperature.　The　as-synthesized　spherical　COF　served　as　an　adsorbent　in　dispersive　solid-phase　extraction　(dSPE),　by　its　virtue　of　great　surface　area　(1542.68　m2/g),　regular　distribution　of　pore　size　(2.95 nm),　and　excellent　stability.　Therefore,　a　simple　and　high-efficiency　dispersive　solid　phase　extraction　method　based　on　a　spherical　COF　coupled　with　high-performance　liquid　chromatography-tandem　mass　spectrometry　(HPLC-MS/MS)　was　established　to　determine　aryl　organophosphate　esters　in　biological　samples.　This　approach　displayed　favorable　linearity　in　the　range　of　10.0–1000.0 ng/L　(r ＞ 0.9989),　a　high　signal　enhancement　factor　(58.8–181.8　folds)　with　low　limits　of　detection　(0.3–3.3 ng/L).　Moreover,　it　could　effectively　eliminate　complex　matrix　interference　to　accurately　extract　seven　aryl　organophosphate　esters　from　mouse　serum　and　tissue　samples　with　spiked　recoveries　of　82.0%–117.4%.　The　as-synthesized　spherical　COF　has　been　successfully　applied　in　sample　preparation.　The　dSPE-HPLC-MS/MS　method　based　on　a　spherical　COF　has　potential　application　to　study　the　pollutants＇　metabolism　in　vivo.　©　2023　Wiley-VCH　GmbH.