We developed a fast and new direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method for the determination of estrone in environmental water based on a novel molecularly imprinted film of controlled thickness as an artificial antibody. The imprinted film was directly synthesized on the well surface of MaxiSorp polystyrene 96-well plate by a room temperature ionic liquid-mediated chemical oxidative polymerization in conjunction with molecular imprinting technology. This novel film was characterized, and results showed that it exhibited an antibody-like binding ability, rapid adsorption speed, high stability, and hydrophilicity, which was particularly advantageous and suitable for BELISA development. This BELISA method had a higher selectivity for estrone than for the structurally related compounds, and competitive binding studies demonstrated various degrees of cross-reactivity with five estrogenic compounds ranging from 30 to 47%. Eighty minutes of analysis time was reduced when compared to that of traditional ELISA, while the imprinted film was able to be reused for more than 50 times without loss of sensitivity. The IC(50) (calculated as the concentration giving 50% inhibition of color development) and the detection limit values under optimized experimental conditions were 200 +/- 40 microg L(-1) and 8.0 +/- 0.2 microg L(-1), respectively. This developed method was applied to the determination of estrone in spiked environmental water samples with excellent recoveries ranging from 80 to 95%, and the results correlated well with that obtained using the high performance liquid chromatography method.