In recent years, the number of reported Human African Trypanosomiasis (HAT) cases caused by Trypanosoma brucei (T.b.) gambiense has been markedly declining, and the goal of ‘elimination as a public health problem’ is within reach. For the next stage, i.e. interruption of HAT transmission by 2030, intensive screening and surveillance will need to be maintained, but with tools and strategies more efficiently tailored to the very low prevalence. We assessed the sequential use of ELISA and Immune Trypanolysis (ITL) on dried blood spot (DBS) samples as an alternative to the traditional HAT field testing and confirmation approach. A cross-sectional study was conducted in HAT endemic and previously endemic zones in Kongo Central province, and a non-endemic zone in Haut Katanga province in the Democratic Republic of the Congo (DRC). Door-to-door visits were performed to collect dried blood spot (DBS) samples on filter paper. ELISA/T.b. gambiense was conducted followed by ITL for those testing positive by ELISA and in a subset of ELISA negatives. In total, 11,642 participants were enrolled. Of these, 11,535 DBS were collected and stored in appropriate condition for ELISA testing. Ninety-seven DBS samples tested positive on ELISA. In the endemic zone, ELISA positivity was 1.34% (95%CI: 1.04–1.64). In the previously endemic zone and non-endemic zone, ELISA positivity was 0.34% (95% CI: 0.13–0.55) and 0.37% (95% CI: 0.15–0.60) respectively. Among the ELISA positives, only two samples had a positive ITL result, both from the endemic zone. One of those was from a former HAT patient treated in 2008 and the other from an individual who unfortunately had deceased prior to the follow-up visit. Our study showed that a surveillance strategy, based on DBS samples and centralized testing with retracing of patients if needed, is feasible in DRC. ELISA seems well suited as initial test with a similar positivity rate as traditional screening tests, but ITL remains complex. Alternatives for the latter, also analyzable on DBS, should be further explored.