Flow injection spectrophotometric analysis (FIA) of human salivary α-amylase was developed using an enzyme degradation reaction of starch–iodine complexes. In this proposed method, the salivary α-amylase, known as a human stress indicator, is directly and rapidly determined without any pretreatment. In this study, the optimum starch–iodine complexes ( i.e. , optimum molecular weight and amylase–amylopectin compounding ratio) were selected, and their rapid degradation in the flow channel was investigated to determine salivary amylase in the FIA system. The determination range of α-amylase was obtained from 0.25 to 5.0 kilo Novo unit per milliliter (KNU/mL), and these concentrations were equivalent to the real concentration of amylase in human saliva. The quantitative values obtained by this method were found to be highly reproducible with 1.6% ( n =25) of the relative standard deviation for 1.0 KNU/mL. The detection limit (3σ) was 60 NU/mL. In addition, the method requires small volume of a sample (20 µL), and 30 samples was sequentially measured within one hour. Real human saliva collected before and after exercise was utilized to demonstrate the feasibility of human stress test and analytical performance of this approach.