A method for the enzymatic determination of adenosine triphosphate (ATP) and glycerol has been developed, using an automated FIA system. The enzymes Glycerophosphate Oxidase (GPO) and Glycerol Kinase (GK) have been immobilized on non-porous glass beads following optimization of the immobilization parameters. The fabricated Single Bead String Reactors (SBSR) were used in connection with a FIA system, following optimization of its parameters. The half-life of GK SBSR, regarding enzyme activity, was found to be 45 days for 90 measurements of daily use and storage at 4°C in the appropriate buffer. The linear dynamic ranges of the working curves for the enzymatic determinations of ATP and Glycerol, were 4–70 μmol.l−1and 2–160 μmol.l−1respectively. Glycerol was determined in white and red wines after a 1:5,100 dilution. Glycerol was also determined in human serum, following deproteinization of the samples by gel filtration. The low limits of quantification (4μmol.l−1ATP and 1 μmol.l−1glycerol) and accuracies better than 2% for ATP and 1% for glycerol, show that the proposed method offers many significant improvements over the conventional methods.