The invention relates to the technical field of biomass detection and discloses an online detection method and device of a hydrothermal pre-treatment process of biomasses. The online detection device comprises a pre-hydrolysis liquid sampling device, a filtering device, a cooling device, a sample preparation device and a spectrum detection device, and a data processing module, wherein the filtering device, the cooling device, the sample preparation device and the spectrum detection device are connected in sequence and are used for purifying a sample; the data processing module is used for acquiring data and calculating a detection model; the sample preparation device is connected with a buffering solution storage device through a buffering solution pipeline; the online detection device is further provided with a metering pump, a flow meter and a solenoid valve, which are used for detecting the flows of pre-hydrolysis liquid and buffering liquid, which flow into the sample preparation device respectively. The online detection method comprises the following steps: carrying out fitting modeling according to obtained absorbance and pre-hydrolysis liquid ratio to obtain a detection model; calculating by utilizing the detection model to obtain a pre-hydrolysis yield and cellulose and hemicellulose removing rates respectively. The device and the method have very good practicability and very high accuracy, and have important practical value on online detection and control of hot water pre-hydrolysis technological effect of enterprises.