Abstract: Addressing the challenge of ineffective traceability in Helicobacter pylori ¹³C urea breath test (13C-UBT) in China, this study employed a dynamic continuous generation system to produce reference gases with delta over baseline (DOB) values ranging from 0 to 50‰. The metrological performance of ¹³C breath test equipment was evaluated using these reference gases. Experimental results demonstrated good agreement between the DOB reference gas values and the measurements of the ¹³C breath test equipment, with indication errors at each concentration point less than ±1.5‰ and repeatability within 0.3‰. To simulate human breath conditions (34°C, 90% RH), reference gases were humidified using a standard gas humidification device. The study compared the performance of ¹³C breath test equipment using reference gases under three conditions: simulated human breath (34°C, humidified), room temperature with humidity, and room temperature without humidification (dry gas). Results showed no significant differences or trends among these conditions, supported by theoretical derivations and principle analyses. This demonstrates that altering the temperature and humidity of reference gases does not affect the reported DOB values. Consequently, room temperature dry gas can be used as a reference gas for calibrating ¹³C-UBT analyzers and similar devices, simplifying the traceability process without compromising accuracy.