We have developed a real-time PCR assay for detection of Trypanosoma brucei DNA in human blood samples. The PCR was conducted with newly designed primers targeting the 177-bp repeat satellite DNA in T. brucei and with Sybr Green to monitor the amplicon accumulation. DNA purification using Chelex 100 resin was performed on blood samples collected on Whatman FTA cards and was shown to be a simple and quantitative method as revealed by real-time PCR. The detection limit of the assay was 100 trypanosomes per mL blood, corresponding to an analytical sensitivity of 0.1 genome equivalents. Trypanosome DNA was detected in all blood samples from sleeping sickness patients and, furthermore, the identity of the amplicon was confirmed in all assays by dissociation analysis. Although template DNA from blood samples was amplified with significantly lower efficiency than genomic DNA, similar efficiency between all assays ensured quantitative results. No amplicon product was obtained with samples from uninfected individuals. The results indicate that the real-time PCR assay described is a rapid and sensitive method suitable for the detection of T. brucei in human blood samples in routine clinical laboratory practice.