The research focuses on the effect of cold plasma treatment on polylactic acid film with different time (0, 15 s, 30 s, 45 s and 60 s), to determine the film morphology and hydrophilicity. Based on that, the polylactic acid films treated with cold plasma were immersed in nisin solution, and the antimicrobial activity was evaluated. The results indicate that cold plasma time can significantly change the hydrophilicity and roughness of the film surface. Moreover, it is found that with the cold plasma time increases, the roughness firstly increases and then decreases, whereas, the water angle initially decreases and then increases. Furthermore, the polylactic acid films with 60 s cold plasma treatment present a better antimicrobial ability against Listeria monocytogenes. Herein, it is concluded that cold plasma treatment can improve the applicability of polylactic acid film in antimicrobial food packaging.