Graphene materials can emit far-infrared ray, but the biological effects of graphene far-infrared ray have not been studied. Furthermore, the non-thermal biological effect of far-infrared ray on organism has not been systematically studied independently of the thermal effect. The purpose of this study was to investigate the non-thermal biological effect of graphene far-infrared ray (gFIR) on Saccharomyces cerevisiae cells. In this work, stringent control of the cultivation conditions was carried out to ensure the stability and constancy of the culture and its temperature. Flow cytometry was used to detect the non-thermal effect of gFIR irradiation on cell membrane permeability, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) content. Compared with the control group, cell membrane permeability of the gFIR exposure cells decreased by 4.7%, MMP increased by 16% and intracellular ROS reduced by 10.7%. The results revealed the valuable features of the non-thermal biological effect of gFIR on Saccharomyces cerevisiae cells, and the further analysis demonstrated that graphene far-infrared materials should have great application value in disease prevention and health promotion.