Trace amounts of copper decrease significantly the oxidative stability of biodiesel while antioxidants, mostly tert-butylhydroquinone (TBHQ), are commonly introduced to the biofuel to guarantee induction period values within regulatory limits. Hence, a simple method to monitor the most used antioxidant and the most deleterious contaminant in biodiesel can give a fast response on its quality. In this context, this work presents a simple, efficient, low cost and sensitive method for the simultaneous and direct determination of copper and the antioxidant TBHQ in biodiesel. The biodiesel samples were diluted in 90% (v/v) ethanol and 10% (v/v) water containing 0.1 mol L−1 HCl (final concentration) as the supporting electrolyte generating a homogeneous mixture in which Cu(II) and TBHQ were directly detected using the square wave voltammetry (SWV) on a gold working electrode. The presence of biodiesel in the selected electrolyte facilitated the separation of analyte peaks facilitating the treatment of data and quantification. The linear responses for the simultaneous determination were in the range between 5 and 100 μg L−1 and 4.2–81.5 mg L−1 for copper and TBHQ (R > 0.997), respectively. High precision (RSD = 4.0% and 3.9%, n = 20) and limit of detection (LOD) values of 0.31 μg L−1 and 1.0 mg L−1 (0.030 μg g−1 and 0.102 mg g−1 in biodiesel) for Cu(II) and TBHQ, respectively, were obtained. Recovery values between 97% and 104% for both copper and TBHQ were obtained. Display omitted •Rapid method for simultaneous determination of copper and the antioxidant TBHQ in biodiesel.•Biodiesel aliquot 100-fold diluted in hydroethanolic electrolyte with 0.1 mol L−1 HCl in the cell.•The presence of biodiesel facilitated the separation of the voltammetric peaks of analytes.•LOD values of 0.030 μg g−1 and 0.102 mg g−1 in biodiesel for Cu(II) and TBHQ, respectively.•Fast protocol to identify metallic contaminants and attest the required presence of TBHQ.