Certain saliva enzymes may be useful biomarkers for detecting exposures to organophosphate pesticides and chemical nerve agents. In this regard, saliva biomonitoring offers a simple and noninvasive approach for rapidly evaluating those exposures in real time. An electrochemical sensor coupled with a microflow injection system was developed for a simple, rapid, and sensitive characterization of cholinesterase (ChE) enzyme activities in rat saliva. The electrochemical sensor is based on a carbon nanotube (CNT)-modified screen-printed carbon electrode (SPE), which is integrated into a flow cell. Because of the excellent electrocatalytic activity of the CNTs, the sensor can detect electroactive species that are produced from enzymatic reactions with extremely high sensitivity and at low potentials. The electrochemical properties of acetylcholinesterase (AChE) enzymatic products were studied using a CNT-modified SPE, and the operation parameters such as the applied potential and substrate concentration were optimized to achieve the best performance. The AChE enzyme activity was further investigated using the CNT-based electrochemical sensor with commercially available purified AChE and ChE in saliva obtained from nave rats. It is found that the calibration curve is linear over a wide range of AChE concentrations from 5 pM to 0.5 nM, and the sensor is very sensitive with the detection limit down to 2 pM. The dynamics of the ChE enzyme activity in saliva with organophosphate pesticides was further studied using this sensor. The results showthatthe senor can be used to characterize salivary enzyme activity and to detect the exposure to organophosphate compounds. This new CNT-based electrochemical sensor thus provides a sensitive and quantitative tool for noninvasive biomonitoring of the exposure to organophosphate pesticides and nerve agents.