Abstract: An effective preconcentration method of hollow fiber based liquid-liquid-liquid microextraction(HF-LLLME)was proposed for the concentration of trace clenbuterol in swine urine and milk samples,and a high performance liquid chromatography with a ultraviolet detector(HPLC/UV)was used for the subsequent detection and quantification. In the process of HF-LLLME,some influence factors including the kind of organic solvent,extraction time,stirring rate and the salt effect were optimized,and toluene was finally chosen as the organic solvent,the extraction time and stirring rate were set as 30 min and 4000 r/min without salt addition in the donor phase,respectively. The analytical performance of the method was studied under the optimal conditions. Results showed a good linearity(r=0.9980)within 1.5~1000 μg/L. The limit of detection(S/N=3)was 0.5 μg/L and the limit of quantitation(S/N=10)was 1.5 μg/L. Compared with a direct injection without HF-LLLME,the high enrichment factor improve the detection sensitivity of HPLC/UV for clenbuterol by about two orders of magnitude. The recoveries at three spiked levels(5,50,500 μg/L)were 87.0%~102.4% with RSD range from 5.1% to 9.8% in swine urine and 80.2%~94.4% with RSD range from 2.7%~6.4% in milk,respectively. The method has advantages such as high enrichment efficiency,good operability,environmental protection,economic and reliability,and was successfully applied to the quantitative analysis of trace clenbuterol in swine urine and milk samples.