The transesterification reaction to produce biodiesel generates a residual content of free glycerol, which may cause several problems to the engines. According to the international specification, the free glycerol determination involves a derivatization step followed by chromatographic analysis, which is done by advanced laboratories. Argentina is one of the largest biodiesel producers in the world, and part of this production is carried out by small and medium-sized producers. For those producers, this analysis is expensive and requires a considerable amount of time because there are few laboratories and they are localized far from the production site. This work proposes an alternative method for free glycerol determination in biodiesel samples taking advantage of automation, the use of an economic and available kit of enzymes, and a rapid and simple detection which was improved by the use of a fit-for-purpose chemiluminescence detection chamber designed in our laboratory. Quantification of free glycerol was performed by chemiluminescence at 425 nm after the reaction of hydrogen peroxide and luminol in the presence of Co2+. The hydrogen peroxide was generated by sequential enzymatic reactions based on the catalytic effect of glycerol kinase and glycerol phosphate oxidase. The calibration curve was linear in the 0.0045-0.0200% (w/w) range (r = 0.9910), and the sample throughput of the automatic system was 8 h(-1). The geometrical configuration of the designed chemiluminescence detection chamber allowed an appropriate mixture of the reagents, due to a coupled efficient mixing system, and a highly sensitive detection (LOD value: 0.0013% (w/w)). The enzymatic method was applied for the determination of glycerol in commercial (soybean) and laboratory syntethized biodiesel samples (corn and sunflower). The trueness of the method was evaluated by comparison with the reference method (EN 14105), and a nonsignificative difference between them was observed (n = 6; P = 0.05).