The purpose of this study was to separate and determine Nystatin in water, urine and plasma samples using a method based on SODIUM dodecyl sulfate (SDS) coated magnetic nanoparticles (nano-magnets Fe3O4) along with spectrophotometry which has been developed for to this end. Due to their excellent adsorption capacity and high chemical stability, nanoparticles can be modified by surfactants. The extraction efficiency of Nystatin can be affected by different factors such as the amount of adsorbent, pH value, eluent type and its volume, extraction time, and ionic strength; these factors have been further studied and optimized. The method was successfully employed for extracting Nystatin from water, urine and plasma samples under optimized condition. The linear response of the method was over ranges of 1-20, 1-18 and 1-15 mg l-1 and the coefficients of determination were 0. 991, 0. 994 and 0. 991; these desirable coefficients were achieved for of water, urine and plasma samples, respectively. In addition, it was tried to investigate the relative recovery in different water, urine and plasma matrices and subsequently the values of 99%, 98% and 102% were obtained. It could be concluded that the method employed here was conveniently fast, linear, efficient and economical for extracting Nystatin in water and biological samples.