An electroenzymatic method based on two coupled enzymatic activities (glycerokinase and glycerol-3-phosphate oxidase) was developed using a platinum microelectrode for the determination of free glycerol in biodiesel samples. The electroenzymatic method proposed showed a good linear correlation coefficient (R = 0.9894), with a linear response in the concentration range of 8.2 x 10(-5) to 5.7 x 10(-4)% (w/w) and a limit of detection of 6.0 x 10(-5)% (w/w). The results obtained for the free glycerol content of select biodiesel samples were compared with their gas chromatography (GC) analyses. The relative errors for glycerol determination using this enzymatic-amperometric method were in the range of -8.0 to 3.0%. The proposed method was shown to be promising for the analysis of glycerol in biodiesel samples and a simple and inexpensive method in comparison to gas chromatography.