The fabrication of nanostructures is of great importance in producing biomedical devices. Significantly, the nanostructure of the polymeric film has a significant impact on the physical and biophysical behavior of the biomolecules. This study presents an efficient nanofabrication method of nanogroove structures on an acrylic film by the micro-embossing process. In this method, a master mold was made from a thermos oxide silicon substrate using photolithography and etching techniques. An isotropic optical polymethyl methacrylate (PMMA) film is used in the experiment. The acrylic film is known for its excellent optical properties in products such as optical lenses, medical devices, and various general purpose engineering plastics. Then, the micro-embossing process was realized to fabricate nanogroove patterns on an acrylic film by using a micro-embossing machine. However, the morphology of the nanopatterns on an acrylic film was characterized by using an atomic force microscope to measure the dimensions of the nanogroove patterns. The impact of embossing temperature on the morphology of nanogroove patterns on acrylic film is experimentally investigated. The results show that when the embossing temperature is too small, the pattern is not fully formed, and slipping occurs in nanopatterns on the acrylic film. On the other hand, the effect of increasing the embossing temperature on the morphology of nanogrooves agrees with the master mold, and the crests between the nanogrooves form straight edges. It should be noted that the micro-embossing temperature also strongly influences the transferability of nanopatterns on an acrylic film. The technique has great potential for rapidly fabricating nanostructure patterns on acrylic film.