The accurate identification of β-lactamases produced by Enterobacteriaceae is a major challenge in clinical laboratories in order to optimize antimicrobial treatment and patient care. We describe here a rapid voltammetric-based method to detect and to discriminate β-lactamase activity in Enterobacteriaceae i.e., penicillinase, cephalosporinase (inducible or overproduced), extended-spectrum beta-lactamase and carbapenemase producers. After a 2-h growth step of the sample under three separate conditions: 1) LB (Luria-Bertani) medium, 2) LB supplemented with 4 μg/mL cefotaxime and 3) LB supplemented with 4 μg/mL cefotaxime and 100 μg/mL potassium clavulanate, the β-lactamase activity was measured by incubating a 0.5 mM nitrocefin solution for 15 min followed by the voltammetric detection of the hydrolyzed nitrocefin with disposable carbon screen printed sensors. The development and the calibration of the method were carried out by analyzing pure cultures of fifty-seven strains with well characterized β-lactam-resistance phenotypes. Thanks to the combination of the three currents (i(1), i(2), i(3)) recorded for each tested bacteria, the proposed procedure allowed to distinguish the different classes of β-lactamase producers. In the second part of the study, the method was applied to the analysis of one hundred and fifteen samples Enterobacteriaceae-positive blood culture samples of bacteraemic patients. Overall data showed that the voltammetric method offered a sensitivity of 100% and a specificity of 80%. Interestingly, all of sixteen samples infected by a third-generation cephalosporins-resistant bacteria (i.e. ESBL and overproduced cephalosporinase producers) were detected. This study clearly demonstrated that the voltammetric assay is an efficient alternative technique for the rapid discrimination of β-lactamases-producing Enterobacteriaceae in blood culture. In contrast to the approved routine assays, the electrochemical test did not require isolated colonies to be performed and was thus carried out in less than 3 h which could allow early administration of an appropriate antibiotic therapy.