A novel calorimetric biosensor is developed by recirculating flow injection analysis. The biosensor consists of a peristaltic pump, two switching valves, a thermostat, an injection valve, two twin cells (a reaction cell and a reference cell), a thermopile, a data acquisition module, and a personal computer (PC). This biosensor with an improved structure can respond to reaction heat timely by placing the thermopile between the reaction and reference cells. Enzymatic reaction will be continuous when an excessive substrate is introduced into the two cells and flows circularly through them. The calorimetric biosensor has been applied successfully in dichlorvos detection by a new test method. In this method, two parameters of constant response in the zero-order reaction phase and the transition moment from zero-order reaction to firstorder reaction are obtained from the progress curve of the enzymatic reaction. Under a certain initial enzyme activity derived from the transition moment, dichlorvos can be determined by comparing the difference in constant response caused by the enzymatic reaction with inhibition in the reaction cell and without inhibition in the reference cell. The performance shows a linear range of 0.01–0.5 mg/L with a relative standard deviation (RSD) of 3–10% and a limit of detection (LOD) of 0.01 mg/L, as well as a detection duration of 20 min. The system is fast, can repeatedly and easily be used, and can improve automation and prevent possible input errors. It is suitable for the rapid detection of pesticides in the field.