The entry of drugs produced into nature causes many environmental problems, which shows the importance of controlling and measuring these drugs more than before. Due to the importance of this measurement, some environmental laboratories are dedicated for improving a method to determine small amounts of fluoxetine as pollutant in water and a biological sample. In this research study, new techniques were applied for solid phase extraction of insignificant amounts of fluoxetine in water samples by carbon nanotubes and its measurement with ultraviolet-visible spectroscopy in biological samples. These techniques are two-phase systems in which donor phases are fluoxetine-containing aqueous samples and acceptor phases are amino-functionalized carbon nanotubes. The experiments were carried out in two stages of extraction from desolate water samples of fluoxetine using methanol as solvent and the desolate samples were taken to UV-Vis spectrophotometer for further analysis. This method is inexpensive, simple and fast, and is consistent with many of the existing machine methods. Extraction parameters such as the effect of desolating organic solvents, pH of donor and acceptor phases, extraction time, desolation time, mixing speed, volume of donor phase and surfactant effect were optimized and quantitative investigations and measurements were done under optimum conditions. The aforementioned techniques have many advantages including short extraction time, low consumption of organic solvents, deleting the effect of previous experiments, low detection limit, and high concentration factor. Concentration factor and detection limit for fluoxetine were found to be 14.3 and 13.6 μg, respectively.