Based on virtual instrument, the on-line moisture content measurement system was designed, and the measurement condition of the JDG-0.2 vacuum freeze-drying machine was considered during the system development. The system can realize real-time data acquisition, calculation and storage. It can also display the weight, moisture content and the rate of the moved water in the samples. Moreover, the change curve of the moisture content and the rate of the moved water can be shown in this system. The result showed that the relative error to the weight of the samples is less than 0.5%, and the absolutely error to the moisture content is less than 0.5%, which achieved the requirement of the on-line monitoring.