Objective To analyze the difference of the carbohydrate antigen 242 (CA242) results measured by Luminex flow cytometry fluorescence immunoassay with different calibrations and different ambient temperatures, so as to evaluate the effect of ambient temperature on the measurement results. Methods Two CA242 samples with different concentrations (median-and high-value) were selected and the determination was repeated at 11 temperature points (18.3, 20.1, 21.5, 22.2, 24.1, 25.0, 25.4, 26.5, 27.4, 28.5, and 30.0℃) for 5 times by Luminex flow cytometry fluorescence immunoassay. The average value of the detection result at the calibration temperature (25℃) was taken as the reference value, and the biases of the detection results at different ambient temperatures were calculated. Forty-nine CA242 specimens covering high-, median-and low-values were examined at 20, 25 and 30℃ by Luminex flow cytometry fluorescence immunoassay, and the results were compared and correlation analysis was carried out. Results The biases of the median-and high-value samples at 18.3℃ were -35.6% and -29.4%, respectively. The difference between the measurement results at 20℃ and 25℃ was significant for the 49 specimens (P<0.001), and there was a linear correlation between them (Y=0.676 9X+0.374 7, R²=0.990 5). The difference of measurement results was significant between 30℃ and 25℃ (P<0.001), and there was a linear correlation between them (Y=0.896 6X+0.227 0, R²=0.999 4). Conclusion Too low or too high ambient temperature has a negative effect on the determination of CA242 by Luminex flow cytometry fluorescence immunoassay. The ambient temperature should be kept relatively constant and controlled within the range of calibration temperature ±4℃.