Liquid-liquid-liquid microextraction(LLLME) coupled with high performance liquid chromatography was developed for the simultaneous determination of three kinds of local anaesthetics,lidocaine,bupivacaine,and tetracaine in human plasma.The effects of stirring rate,extraction time,and pH in the donor phase on the enrichment factor of analytes were investigated.The optimal experimental conditions,200 μL benzene as organic phase,1.0 μL 0.2 mol/L HCl as acceptor phase,250 r/min of the stirring rate,and 45 min of the extraction time,were selected.Under these conditions,high enrichment factors(more than 305 fold for all analytes) were obtained.The linear range of studied analytes were from 0.025 to 5 mg/L except tetracaine which was from 0.05 to 5 mg/L with correlation coefficients(r) better than 0.996.The limits of detection was 0.005 mg/L for lidocaine,0.015 mg/L for bupivacaine,and 0.025 mg/L for tetracaine at(signal-to-noise) ratio of 3(S/N=3).The relative standard deviations were lower than 5%.With LLLME,some interfering effects from the substances in plasma which interfered with the determination of analytes were eliminated efficiently.The method with little solvent consumption was sensitive and suitable for the determination of lidocaine,bupivacaine and tetracaine in human plasma.This method was applied to the specimen of patient who was treated with anaesthesia of lidocaine and bupivacaine,and results showed the possibility for the simultaneous determination of above two local anaesthetics in human plasma.