The invention discloses a non-plug-in water content sensor of a culture substrate. The non-plug-in water content sensor comprises a unilateral sensitive capacitive probe, a circuit mainboard with a capacitance measurement circuit, and an input/output interface cable, wherein the unilateral sensitive capacitive probe comprises a driving pole plate, induction pole plates, a shield pole plate and a substrate; the driving pole plate and the induction pole plates are positioned on the same surface of the substrate; the shield pole plate is positioned on the other surface of the substrate; and the induction pole plates and the shield pole plate are equipotential. The invention further discloses a calibration model of the sensor. A parameter beta in the calibration model is determined according to a water content output voltage signal of the sensor in a main substrate and a water content output voltage signal of the sensor in a given culture substrate at any point, and a calibration equation is determined sequentially. The sensor can obtain the water content of a surface layer of the culture substrate, and has the advantages of low cost, stable performance, simple input/output interface, convenience and simplicity in calibration, and the like; and the calibration model of the sensor and a two-point calibration model method based on the model have the characteristics of quickness and simplicity.