Betamethasone acetate (BA) is a glucocorticoid steroid with anti-inflammatory and immunosuppressive properties which can be used in treatment of asthma and itching. In this research, polyvinyl alcohol nanospheres loaded by betamethasone acetate were prepared by oil in water emulsion method after which they were exposed to 1, 2 and 4 cycles of freeze-thawing (F-T) process including 24 hours freezing cycle at -25 °C and 24 hours thawing at ambient temperature. Nanospheres fabrication was confirmed by transmission electron microscopy and betamethasone release was analyzed by UV spectrophotometer at 245 nm. The results revealed that by increasing the number of F-T cycles the rate of drug release decreased. The effect of BA concentration was also investigated on human chondrosarcoma (sw 1353). The MTT assay was utilized to assess the cell proliferation. The results showed that these biocompatible nanospheres can be used for sustained release of such drugs for more than four months and drug release rate can be effectively controlled by implementing F-T cycles.