Antibiotics are widely used in dairy cattle production, however this can pose a public health threat, particularly when antibiotic residues remain in the food products like milk. Therefore, new analytical solutions aiming the determination of antibiotics, like the still commonly used penicillin, are welcomed. This work describes a novel electrochemical biosensor, with amperometric and differential pulse voltammetric detection, based on a modified carbon paste with β-lactamase for the determination of penicillin G, a β-lactam antibiotic. It is a 'second-generation' biosensor since cobalt phthalocyanine was used as an electron mediator. The proposed biosensor, by differential pulse voltammetry, presented a limit of detection of 0.079 μmol/L. Using this biosensor, a value of 43 ± 3 μmol/L was determined for the Michaelis-Menten enzymatic constant for penicillinase extracted from Bacillus cereus.