In this work, an electroenzymatic methodology based on two coupled enzymatic activities (glycerokinase and glycerol-3-phosphate oxidase) was developed using an oxygen Clark-type electrode for the determination of free and total glycerol in biodiesel samples. The enzymatic conversion of glycerol consumes oxygen, which is measured amperometrically in a Clark-type electrode and correlated with the concentration of glycerol in the sample. The electroenzymatic method proposed showed a good linear correlation coefficient (R ¼ 0.9990) with a linear response in the concentration range of 6.25 Â 10 À5 to 6.25 Â 10 À4 % (w/v) and limits of detection and quantification at 1.0 Â 10 À5 % and 3.0 Â 10 À5 % (w/v), respectively. Good correlations were found between the results obtained in this work and those by the gas chromatography technique (R ¼ 0.9994). The proposed method was shown to be promising for the analysis of glycerol in biodiesel samples, with a simple and inexpensive methodology compared with the gas chromatography technique.