A three phase hollow fiber membrane microextraction/ultrahigh performance liquid chromatography（HF-LPME/UPLC） was developed for the determination of eight amino acids in Escherichia coli..The three phase hollow fiber membrane micro extraction system was designed.The extraction conditions for HF-LPME were optimized as follows:the supported liquid membrane:n octanol,the concentration for NaOH in receiving phase:030 mol/L,the concentration for hydrochloric acid in sample solution:50 mmol/L,the concentration for NaCl:200 g/L,the extraction temperature:25 ℃,the stirring speed:500 r/min and the extraction time:4 hours.Eight kinds of amino acids were extracted after derivatization,and finally analyzed by UPLC.A baseline separation was achieved within 5 min.The calibration curves for the analytes were linear in range of 020-49×103 μmol/L with their correlation coefficients（r2） more than 0999,the enrichment factors of 110-290 and the detection limits of 00l/L.At a spiked concentration of 10 μmol/L,the recoveries for eight amino acids in Escherichia coli.were 887%-1031% with the relative standard deviations of 32%-43%（n=5）.The proposed method was highly sensitive and reproducible,and could be used for the detection of amino acids in bacteria.