Abstract Flame stability of premixed biogas flame for Reference Test Burner (RTB) was investigated. In this study, six kinds of test gases were used to simulate biogas in which CO2 volume fraction varied from 30% to 45%. A series of experiments were conducted on two RTBs with different port diameters and at different outlet unburned mixture temperatures. It was found that the lifting and yellow tipping limits show similar trends regardless of the biogas components, port diameters and mixture temperatures. A “float off” phenomenon could be observed at low gas flow rate and low primary air ratio. Low mixture temperature, small ports and high CO2 concentration in biogas can lead to the unstable condition of “float off”. The lifting limits are enhanced with an increase of port diameter or mixture temperature and with a decrease of CO2 concentration. The yellow tipping limits are extended with an increase of CO2 concentration and with a decrease of mixture temperature or port diameter. In addition, the lifting limit curve becomes a straight line when semi-logarithmic graph paper is applied. The intercept increases with a decrease of the CO2 concentration in biogas and with an increase of port diameter or gas temperature.