Glucose oxidase (GO) was entrapped in an electrodeposited poly(tyramine) film together with a negatively charged sulfobutylether-beta-cyclodextrin (SBCD) on the active area of a platinum (Pt) nanoparticle modified boron-doped diamond (BDD) electrode. Electrodeposition of tyramine and simultaneous entrapment of GO/SBCD were performed in 50 mM phosphate buffer, pH 7 containing 0.1 M tyramine, 1750 units GO, and 10 mM SBCD. Atomic force microscopy (AFM) imaging revealed the presence of semicircular nanofibers with a height of 40 nm and an averaged length of 795 nm throughout the electropolymerized film surface. The combined film of poly(tyramine) and SBCD served as an excellent matrix polymer for the GO immobilization with high stability, selectivity, and reproducibility. Sensitive and selective detection of H2O2 was realized at +0.4 V vs 3 M Ag/AgCl, since the poly(tyramine)/SBCD film was capable of preventing the passage of electroactive uric and ascorbic acids to the electrode. The glucose biosensor exhibited a remarkably selective response to glucose with a detection limit of 10 mu M, linearity up to 110 mM, and a response time of 2 s. Glutaraldehyde cross-linking of the film with entrapped GO completely eliminated electroactive interference caused by uric and ascorbic acids..