Three-dimensionally ordered polyaniline (PANI) doped with poly (styrene sulfonate) inverse opal was fabricated by electrochemistry. The as-synthesized porous PANI composite film on the platinum electrode used colloidal crystals of silica beads as sacrificial template. The poly(diallyldimethylammonium chloride) (PDDA)/glucose oxidase (GOx) multilayer [(PDDA/GOx)(n)] was self-assembled in the synthesized ordered porous PANI for biosensing applications. The inverse opal of PANI architecture offered a microenvironment for increasing the quantity of GOx. The excellent electrocatalytic activity of (PDDA/GOx)(3)/PANI/Pt toward glucose and special three-dimensionally ordered structure of the enzyme electrode resulted in good characteristics, such as a high sensitivity (64.4 mu A mM(-1) cm(-2)) and a short response time (within 5 s). (c) 2007 The Electrochemical Society.