2,4,6-Trinitrotoluene (TNT) and its four metabolites, namely 2-ADNT, 4-ADNT, 2,4-DANT, and 2,6-DANT, are highly toxic substances. These metabolites also serve as biomarkers for assessing the health of individuals exposed to TNT. In this study, a homemade DDT-IMS apparatus was utilized to detect these metabolites. Under negative detection mode, the drift times of 2-ADNT and 4-ADNT showed subtle shifts within a drift tube temperature range of 100 °C-120 °C, aiding in their differentiation. In positive detection mode for 2,4-DANT and 2,6-DANT, significant variations were observed in both the number and drift time of their positive product ions across a drift tube temperature range of 80 °C-120 °C. Consequently, optimal analytical performance for these metabolites was achieved at approximately 100 °C. Evaluation of the instrumental response during the measurement of the four metabolites in both positive and negative modes revealed that negative detection mode offered greater advantages of detecting these compounds. The working ranges for measuring the four metabolites spanned two orders of magnitude, with detection limits for each metabolite nearly below 1 ng. Notably, clear identification of the signals for these metabolites was achieved even when samples were mixed in urine, highlighting the ability of the DDT-IMS in detecting TNT metabolites. The developed DDT-IMS detection method has significant potential for enhancing environmental risk assessment and biological hazard evaluation, particularly in relation to human exposure to TNT.