Closed soilless substrate cultivation is the direction of modern agricultural transformation, substrate water content is an important detection indicator of the cultivation process, but the study of the soil moisture sensor used for detecting water content of substrate is rear. To solve this problem, this paper evaluated the performance of a frequency domain soil moisture sensor FDS-100 in the substrate water content detection comprehensively. For different proportions of the substrate, compared and analyzed the standard water content obtained from the drying method and FDS-100 measurement. Besides, it also compared the measure performance of FDS-100 and ECH2O-5TE in a mixed substrate. The influence of compaction degree, EC value and temperature value on sensor is also tested. The results shows that measured values from FDS-100 measurement in different proportions substrate did not follow the same curve;the degree of compaction impacted the FDS-100 measurement, measured value increased with the degree of compaction, the measurement error is between -1% and 10%;EC had a certain influence on FDS-100 measurement, measurement error is between ±5%;temperature had less effect on the measurement of FDS-100, the measurement error is below 2%;the relative error between FDS-100 and ECH2O-5TE is 4.32%. In summary, FDS-100 can be used in substrate moisture detection, it should be calibrated for different substrates and different degrees of compaction in actual use.