It is considered that measurement of salivary α-amylase activity is a useful tool for evaluating the sympathetic nervous system. The purpose of this research is to demonstrate a new design of a flat-chip microanalytical enzyme sensor (flat-chip sensor) for salivary amylase activity as a Micro-Electro-Mechanical Systems (MEMS), which may be used for wearable analytical systems. To meet this purpose, the biosensor needs to be miniaturized and to possess high-sensitivity. A pre-column and a flat-enzyme electrode were incorporated in a flow cell of volume 25.7 ml. In order to miniaturize the flow cell, two enzymatic membranes containing maltose phosphorylase obtained from Enterococcus hirae (MP membrane) and glucose oxidase and peroxidase (GOD-POD membrane) were immobilised on the same planar surface. As a result, a flat-chip sensor incorporating a flow cell as small as a C battery was produced. The optimum conditions of three parameters of the fabricated flat-chip sensor, the immobilising method of the enzymatic membrane, dropping volume of the mixed enzymatic solution and flow rate of the sample solution, were examined. An analytical system for 0–190 kU/l amylase activity with R2 of 0.97 was fabricated with a sample volume of 50 μl. This research indicates the possibility of a wearable biosensor for continuous monitoring of salivary amylase activity.