After decades of development, the tunable diode laser absorption spectroscopy (TDLAS) became one of the most promising techniques for online trace gas analyzing in process industry. Limited by its principle, the measurement result of TDLAS system is seriously affected by temperature and gas pressure variation. For this reason, most TDLAS systems employed temperature and pressure sensors, which can provide information for partly correcting the error. Theoretically, the gas absorption theory itself is not perfect enough to give an analytical relation between the measurement error and the temperature & pressure variation. Practically, temperature and pressure sensors are not available in some harsh working condition. To address these problems, an online self-calibration technique with a reference gas cell is proposed to compensate temperature and pressure variation induced measurement error in a TDLAS system. More specifically, a reference gas cell filled with known proportion target gas is placed on site, surrounded by working gas to be measured. The main body of the gas cell is made from a stainless tube, one end is a silica glass window and the other end is a reflector. A pressure bellows is connected to the middle of the stainless tube by a branch conduit. The pressure bellows can adaptively deform to keep the pressure balance between the inside and outside gas. Thereby, the temperature and pressure inside the reference cell are equal to that of the gas outside. To ensure the similarity between the reference gas cell and working gas cell, they share the same laser diode source and signal processing circuit. In one working cycle, the TDLAS system obtains the absorption spectrum of both gas cells synchronously. Then the concentration of the trace gas can be easily obtained by calculating the absorption intensity proportion of both absorption spectra without considering the affection of temperature and pressure. The principle, design, and experiments of this technique were presented in this paper.