The main aim of this research is using the preconcentration and sample preparation method, based on directly suspended drop microextraction method of amitriptyline prior to UV-vis spectrophotometer. Here, the target compound was extracted from an aqueous sample solution (donor phase) into an organic solvent drop (acceptor phase), which was suspended in the sample solution without using a microsyringe needle as a supporting device. Several important parameters which influence the extraction efficiency such as the kind of extracting solvent, extraction time, volumes of the sample solution and organic extraction solvent, stirring rate, pH of the donor phase and salt effect were investigated. Under the optimal conditions the enrichment factor was 25. By plotting absorbance versus concentration of the analyte in the standard solutions, calibration curve was obtained with correlation coefficient of 0.9931. The linearity of the method has been investigated between the ranges of 0.04-1 μg mL-1. The Limit of Detection (LOD) of the method which calculated theoretically was 0.013 μg mL-1. The precision or repeatability of the method is based on the average Relative Standard Deviation (RSD%) is 4.6, for three different concentrations of the analyte. Finally, the proposed method was applied for the determination of amitriptyline in urine and environmental water samples and the reasonable relative recoveries were obtained.