We developed two glucose biosensors based on poly(3,4-ethylenedioxythiophene) (PEDOT) and polyacrylic acid (PAA) doped with poly(4-lithium styrenesulfonic acid) (PSSLi) or PEDOT and anthranilic acid (AA) doped with poly(4-styrenesulfonic acid) (PSSH). The first one was already mentioned in literature in similar configurations; however, the second one has not been described before. The glucose oxidase was immobilized by means of covalent bonding, which has been proved by spectrophotometric assay with o-dianisidine. Amperometric measurements were carried out for the determination of glucose in phosphate buffer. The prepared electrodes functioned as first-generation glucose biosensors at applied potential E = + 0.6 V during t = 30 s. The elaborated procedure was validated, and all of the statistical parameters met the requirements for biosensors. The presented method was successfully applied for determination of glucose concentration in food samples, such as grape juice and honey. The activity of the presented biosensors was also studied in the presence of ascorbic acid and uric acid, acting as interfering agents.