Resonate frequency and cell constant of photoacoustic spectrum system are usually calibrated by using standard gas in laboratory, whereas the resonate frequency and cell constant will be changed in-situ, leading to measurement accuracy errors, caused by uncertainties of standard gas, differences between standard and measured gas components and changes in environmental condition, such as temperature and humidity. As to overcome the above problems, we have proposed an on-line atmospheric oxygen-based calibration technology for photoacoustic spectrum system and used in measurement of concentration of carbon dioxide in atmosphere. As the concentration of atmospheric oxygen is kept as constant as 20.96%, the on-line calibration for the photoacoustic spectrum system can be realized by detecting the swept-frequency and peak signal at 763.73 nm. The cell of the PAS has a cavity with length of 100 mm and an inner diameter of 6 mm, and worked in a first longitudinal resonant mode. The influence of environmental temperature and humidity, gas components on the photoacoustic cell’s performance has been theoretically analyzed, and meanwhile the resonant frequencies and cell constants were calibrated and acquired respectively using standard gas, indoor air and outdoor air. Compared with calibrated gas analyzer, concentration of carbon dioxide is more accurate by using the resonant frequency and cell constant calculated by oxygen in tested air, of which the relative error is less than 1%, much smaller than that calculated by the standard gas in laboratory. The innovation of this paper is that using atmospheric oxygen as photoacoustic spectrum system’s calibration gas effectively reduces the error caused by using standard gas and environmental condition changes, and thus improves the on-line measuring accuracy and reliability of the photoacoustic spectrum system.