A new fluorometric method was developed for the determination of alpha-amylase activity in human serum samples. Firstly, a saturated starch-iodine complex (SI) was prepared. The SI complex was combined with sodium fluorescein to form a starch-iodine-sodium fluorescein complex (SIF). As the SIF complex decomposes with the alpha-amylase enzymatic hydrolysis of starch, the intensity of its fluorescence emission increases. The alpha-amylase activity is determined using the increased fluorescence emission intensity following hydrolysis of the SIF complex by alpha-amylase. The optimum pH, optimum buffer concentration, optimum temperature, and interference effect were identified for the developed fluorometric measurement method. Under the optimum conditions, a linear calibration curve was obtained between 0.18 and 9.00 U/L for alpha-amylase. The alpha-amylase activity in the human serum sample was also determined by our prepared measurement system and compared with the result from a medical center. Both methods are in good agreement with each other. Because this newly developed fluorometric method for alpha-amylase activity in serum samples is inexpensive, easy to use, and carried out to detect a very low amount of human serum alpha-amylase with sensitivity, it can be proposed this method for alpha-amylase activity assay in all other biological samples.