In this study, poly(acrylic acid) and multiwalled carbon nanotubes (PAA/MWNTs) composite nanofiber thin film was prepared on fluorine doped indium tin oxide?coated glass substrate through electrospinning method. The electrospun PAA/MWNTs nanofiber thin film was modified with layer?by?layer (LbL) self?assembled multilayers of glucose oxidase (GOD) and poly(dialyldimethylammoniumchloride) (PDADMAC) prior to employ for glucose detection. The obtained nanofiber thin film was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The prepared nanofibers thin film shows a good potential for detection of glucose at constant applied potential through an electrochemical method.