Objective To investigate the effect of temperature and humidity on detection of Treponema pallidum antibodies via high-throughput ELISA detection system. Methods Two high-throughput ELISA detection systems from different manufacturers were selected for performance verification. Daily detection of fixed value quality weak positive control materials was used to qualify these two systems. Temperature, humidity and S/CO values were recorded and data were used for statistically analyze. Results For Addcare ELISA 600 detection system, the univariate analysis of variance of weak positive control values (S/CO) showed a significant difference in either temperature group (F = 6.05, P = 0.0001) or humidity group (F = 3.23, P = 0.0043). At the same humidity conditions, temperature increased by 1 ℃, the S/CO increased 0.043. While under same temperature, when the humidity increased 1%, S/CO value reduced 0.007. For Freedom evolzyer detection system, the univariate analysis of variance of weak positive control values (S/CO) also showed a significant difference when grouped by temperature (F= 30.27, P<0.0001) or by humidity (F=11.99, P<0.000). At the same humidity conditions, temperature increased by 1 ℃, the S/CO value increased 0.037. While under same temperature, when the humidity increased 1%, S/CO value reduced 0.005. Conclusion No significant difference was captured between detection value and the expected value when temperature was 18-22℃，while humidity was 20%-29%, or 30%-39%.The expected value was significantly different with detected value when temperature range were 8-12℃ or 13-17℃, while the humidity was 10%-19%. There is a positive correlation between temperature and the S/CO values, while it’s a positive correlation for humidity and S/CO values.