Methods for the construction of electrochemical composite biosensors using gold nanoparticles and Teflon as nonconducting-binding material are described in detail. The advantages of the incorporation of gold nanoparticles to the composite electrode matrices are highlighted, giving rise to bioelectrodes with improved analytical performance in terms of stability and sensitivity with respect to other biosensor designs. Three different biosensors have been considered: a tyrosinase biosensor in which the enzyme and gold nanoparticles are incorporated into graphite—Teflon composite electrode matrices by simple physical inclusion, a progesterone immunusensor in which the antibody is directly attached to the electrode surface and amperometric transduction is carried out at a colloidal gold—graphite—Teflon—tyrosinase composite biosensor, and a mediator-less glucose oxidase biosensor constructed by bulk incorporation of the enzyme into colloidal gold-multiwall carbon nanotubes—Teflon composite electrodes.