The purified anti-diniconazole antibody was polymerised to hydrolytic tetramethoxysilane (TMOS) to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to diniconazole. The optimized conditions of the IAC were as follows: water as equilibrium and adsorbent medium, 30% and 50% (v/v) methanol aqueous solutions as eluents. The results showed that the dynamic column capacity was up to 125.4 microg/g of bed volume. The river water and fruit samples spiked with diniconazole were cleaned up and enriched by the IAC, and the diniconazole in eluant was determined by high performance liquid chromatography. The average recoveries (n = 5) of diniconazole in river water sample were 90.36%-100.14% with relative standard deviations (RSDs) of 2.03%-6.08%, and the average recoveries in fruit samples were 85.55%-94.02% with RSDs of 3.38%-6.78%. The IAC cleanup procedure provided an effective pretreatment method for the determination of diniconazole in sample media such as river water and fruits.