In the present research, a novel microextraction method was used based on dispersive liquid–liquid microextraction using low‐density solvent for the determination of trace amounts of amitriptyline and doxepin as model compounds in human plasma and spring water samples before gas chromatography and flame ionization detection. The parameters influencing the extraction efficiencies including type of the extracting solvent, volume of the aqueous sample solution (donor phase), volume of the extraction solvent (acceptor phase), the number of air‐injection, and the effects of pH and ionic strength were optimized. Under the optimized conditions, the obtained enrichment factors were >80. By plotting peak areas of the sample solutions versus various concentrations of the analytes, calibration curves were obtained which show the linear ranges of 10–3000 ng/mL with correlation coefficients of >0.996. The precision of the method was calculated <4.2 and the limit of detection was 1 ng/mL for two analytes. The proposed microextraction method was used for the extraction of the analytes in environmental water and plasma samples and the calculated relative recoveries were all >91%.