Direct ethanol fuel cells are attracting much attention due to their excellent performance. Electro-oxidation of ethanol is not a combustion process, which involves multiple reaction processes. The low C—C bond cleavage ability and the poisoning caused by ethanol oxidation intermediate C1 molecules adsorb on the surface of catalysts are bottlenecks which restrict its application. Electrochemical in-situ fourier transform infrared spectroscopy (in-situ FTIRS) is to collect the vibration information of the specific functional groups of the reaction species in situ and reveal the reaction process at the molecular level, helping to understand the reaction mechanism. High performance PtRh/RGO catalyst was used, to investigate the electro-oxidation of ethanol at different temperatures through the combination of isotope tracer and electrochemical in-situ FTIRS. Cyclic voltammetry studies revealed that the electro-oxidation properties of ethanol and the selectivity of C—C bond cleavage ability decreased in the order of: PtRh/RGO (45 ℃)>PtRh/RGO (25 ℃)>commercial Pt/C. Electrochemical in-situ FTIRS revealed the electro-oxidation process at the molecular level. It was found that CO2, CO, —CH3 and —CO characteristic bands increased gradually with the increase of potential. CO2 and CH3COOH are the products of complete oxidation and incomplete oxidation of ethanol, respectively. Therefore, the ratio of the integrated area of the characteristic bands in the infrared spectrum ［CO2］/［CH3COOH］ is the measurement of CO2 selectivity. The band at 1 280 cm-1 was used to quantitatively calibrate CH3COOH, but for the infrared spectra of PtRh/RGO catalyst, the superposition of band CH3COOH at 1 280 cm-1 and methanol derivative appeared at 1 214 cm-1. The superposition band subtraction method was developed to calculate the CO2 selectivity of PtRh/RGO. The selectivity of CO2 on PtRh/RGO at 45 ℃ was improved compared with that at 25 ℃, increased 48.1% at 0.3 V, slightly increased at 0.5 and 0.6 V, but decreased at 0.4 V, which might ascribe to the competitive adsorption of β-C in ethanol and —OH in water. At both reaction temperatures, CO2 selectivity show a downward trend at potentials above 0.4 V. To further investigate the complete oxidation of CO2 derived from α-C or β-C, isotopically-labeled 13CH312CH2OH was used as the probe molecule, combined with electrochemical in-situ FTIRS to study the electro-oxidation of ethanol on PtRh/RGO electrodes at 25 and 45 ℃. The results show that the initial potential of complete oxidation of β-C is independent of temperature, both of which are 0.3 V. By quantitatively analyze the ratio of the integrated area of 13CO2/12CO2, it was found that the ratio under 0.3～0.5 V at 45 ℃ increased 0.11, 0.18 and 0.22 compared with that at 25 ℃, which indicated that the selectivity of β-C increased with the increase of temperature or potential.