The combined presence of autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA) and to the islet-specific cation efflux transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss of immune tolerance and the clinical manifestation of autoimmune diabetes mellitus (DM). The screening of GADA and ZnT8A could be an appropriate alternative to identify diabetic subjects with underlying autoimmunity, helping to reach to a correct diagnosis and guaranteeing the start of an early and adequate treatment. Herein, we describe the expression of a chimera molecule including immunodominant regions of the antigens ZnT8 and GAD65 by using the baculovirus-insect cells system, yielding 30 mg/L culture medium. This recombinant chimera retains the immunoreactive conformation of the epitopes that are recognized by their specific antibodies, so it was used for the development of a high sensitivity (75.51 %) and specificity (98.04 %) bridge ELISA for the detection of highly prevalence ZnT8A and/or GADA, in a one-step screening assay. This immunoassay is useful either to confirm autoimmune diabetes or for detection in routine screening of individuals at risk of autoimmune DM. As DM is a slow progress disease, remaining asymptomatic for a long preclinical period, serological testing is of importance to establish a preventive treatment.