In this paper, a high-resolution laser absorption spectrometer is developed using a distributed feedback (DFB) diode laser in the spectral region near 760 nm, which is designed for the highly sensitive detection of oxygen (O2) in breathing gas. To investigate the spectrometer performance, O2 absorption line at 13146.580 cm−1 was selected, and detailed experiments on air-broadening coefficient, self-broadening coefficient and spectral line intensity were conducted. Compared to the corresponding data listed in the HITRAN2020 database, the results show that the errors for air-broadening coefficient, self-broadening coefficient and line intensity, were −6.832%, 4.082% and 4.667%, respectively. Finally, the developed laser absorption spectrometer was primarily evaluated for real-time analysis of O2 content in human breath analysis. The results indicate that the measured O2 concentrations in several volunteers range from 16% to 16.5%, which is in agreement with the levels reported in the literature for healthy humans.