Real-time measuring of CO2 isotopes (13CO2, 12CO2, and 18OC16O) in exhaled breath using a mid-infrared hollow waveguide gas sensor incorporating a 2.73 μm distributed feedback laser was proposed and demonstrated for the first time based on calibration-free wavelength modulation spectroscopy. The measurement precisions for δ13C and δ18O were, respectively, 0.26 and 0.57‰ for an integration time of 131 s by Allan variance analysis. These measurement precisions achieved in the present work were at least 3.5 times better than those reported using direct absorption spectroscopy and 1.3 times better than those obtained by calibration-needed wavelength modulation absorption spectroscopy. Continuous measurement of three isotopes in the breathing cycle was performed. Alveolar gas from the expirogram was identified, and the 13C/12C and 18O/16O ratios were found to be almost constant during the alveolar plateau, which enables optimization of breath sampling and provides accurate information on metabolic processes. The 13C/12C and 18O/16O isotope ratios at the alveolar plateau of five breath cycles were averaged, yielding δ13C and δ18O values of (−24.3 ± 3.4) and (−30.7 ± 2.6) ‰, respectively. This study demonstrates the feasibility of real-time analysis of 13C- and 18O-isotopes of human breath CO2 in clinical applications and shows its potential for diagnosing respiratory-related diseases with high sensitivity, selectivity, and specificity.