A variety of active substances can be produced during discharge in liquid, in which hydroxyl radical (OH), hydrogen radical (H) is considered as main active species leading to liquid chemical reaction. However, it is difficult to measure them due to its characteristics of short activity and short life; especially it is more difficult to conduct quantitative measurement due to the lack of standard samples. In contrast to the indirect measurement of direct capture, the optical method of measuring free radicals is a direct measurement method, which is characterized by instantaneous on-line measurement that allows immediate data acquisition and time and spatial distribution measurements. In order to study the free radical properties of microwave discharge in water, the active substances produced by microwave discharge were investigated by optical emission spectroscopy. The effects of microwave power and internal pressure on the relative spectral intensities of OH radicals were investigated. The spatial distribution of OH radicals in the plasma was observed. At the same time, the electron excitation temperature was estimated. The experimental results showed that a large amount of OH, H, O radicals could be produced by microwave discharge in water, in which OH radicals had the strongest relative spectral intensity and showed a tendency to increase with the increase of microwave power; but were rapidly weakened with the increase of internal pressure. Free radicals based on OH radicals were mainly produced in the vicinity of the tip of the electrode. At the same time, the microwave excitation temperature of the microwave plasma in water was about 0.33×104 K.