A laser frequency locked hollow waveguide (HWG) gas sensor is demonstrated for simultaneous measurements of three isotopologues (12CO2, 13CO2, and 18OC16O) using wavelength modulation spectroscopy (WMS) with a 2.73 μm distributed feedback (DFB) laser. The first harmonic (1f) signal at the sampling point where the peak of the second harmonic (2f) signal located was employed as locking point to lock the laser frequency to the transition center of 13CO2, while the absorption lines of 12CO2 and 18OC16O were being scanned. Continuous measurements of three isotopologues of 4.7% CO2 samples over 103 minutes under free running and frequency locking conditions were performed, respectively. The measurement accuracy and precision of the three isotopologues achieved under frequency locking condition were at least 3 times and 1.3 times better than those obtained under free running condition. The Allan variance plot of the developed laser-locked HWG gas sensor shows a detection limit of 0.72 ‰ for both δ13C and δ18O under the frequency locking condition with a long stability time of 766 s. This study demonstrated the high potential of a novel human breath diagnostic sensor for medical diagnostic with high accuracy, precision, sensitivity, and without frequently repeated calibration.