Previous research revealed that isotopes ¹³C and ¹⁸O of exhaled CO₂ have the potential link with Helicobacter pylori; however, the ¹⁷O isotope has received very little attention. We developed a sensitive spectroscopic sensor for simultaneous δ¹³C, δ¹⁸O, and δ¹⁷O analysis of human breath CO₂ based on mid-infrared laser direct absorption spectroscopy with an interband cascade laser (ICL) at 4.33 μm. There was a gas cell with a small volume of less than 5 mL, and the pressure in the gas cell was precisely controlled with a standard deviation of 0.0035 Torr. Moreover, real-time breath sampling and batch operation were achieved in gas inlets. The theoretical drifts for δ¹³C, δ¹⁸O, and δ¹⁷O measurement caused by temperature were minimized to 0.017‰, 0.024‰, and 0.021‰, respectively, thanks to the precise temperature control with a standard deviation of 0.0013 °C. After absolute temperature correction, the error between the system responded δ-value and the reference is less than 0.3‰. According to Allan variance analysis, the system precisions for δ¹³C, δ¹⁸O, and δ¹⁷O were 0.12‰, 0.18‰, and 0.47‰, respectively, at 1 s integration time, which were close to the real-time measurement errors of six repeated exhalations.