A rapid, simple and selective method based on molecularly imprinted, spin column extraction coupled with fluorescence detection was successfully established for the determination of 2,4-dinitrophenol in serum samples. The 2,4-dinitrophenol imprinted polymers exhibited highly selective recognition for the template molecule and the maximum adsorption capacity was 138.9 mg/g. The results indicated that when water is used as the loading solution, only 2,4-dinitrophenol could be adsorbed on the spin column without the remaining structural analogs(2-nitrophenol, 4-nitrophenol and phenol). After eluting with acetonitrile/acetic acid(9/1, v/v), 2,4-dinitrophenol in serum samples could be determined by using the fluorescence spectrometer, based on the fluorescence enhancement of fluorescein by the template molecule. Under the optimal conditions, the spiked recovery ranged from 95.8% to 103.4% and the detection limit was 1 nmol/L. The results confirmed the reliability and practicality of the protocol and revealed a good perspective of this method for biological sample analysis. A rapid, simple and selective method based on molecularly imprinted, spin column extraction coupled with fluorescence detection was successfully established for the determination of 2,4-dinitrophenol in serum samples. The 2,4-dinitrophenol imprinted polymers exhibited highly selective recognition for the template molecule and the maximum adsorption capacity was 138.9 mg / g. The results indicated that when water is used as the loading solution, only 2,4-dinitrophenol could be adsorbed on the spin column without the remaining structural analogs (2-nitrophenol, 4- nitrophenol and phenol. After eluting with acetonitrile / acetic acid (9/1, v / v), 2,4-dinitrophenol in serum samples could be determined by using the fluorescence spectrometer, based on the fluorescence enhancement of fluorescein by the template molecule . Under the optimal conditions, the spiked recovery ranged from 95.8% to 103.4% and the detection limit was 1 nmol / L. The results confirmed the reliability and practicality of the pro tocol and revealed a good perspective of this method for biological sample analysis.