Objectives: To develop a novel assay that uses branched DNA technology to measure TMPRSS2-ERG fusion, as genetic rearrangement of TMPRSS2 regulatory sequences and coding sequences of the ERG gene has been detected in nearly half of prostate cancers, but quantitative assays to detect such TMPRSS2-ERG gene fusion have been limited to real-time polymerase chain reaction (PCR) techniques that rely on reverse transcriptase-based amplification. Methods: Branched DNA probes were designed to detect TMPRSS2-ERG gene fusion in prostate cancer cell lines. Nonquantitative nested reverse transcription (RT)-PCR and fluorescence in situ hybridization (FISH) were used to ascertain TMPRSS2-ERG gene fusion status in prostate tissues. Results: The branched DNA assay detected TMPRSS2-ERG gene fusion from less than 200 pg of prostate cancer RNA, whereas more than 600 pg of RNA was required for fusion gene detection by one step real-time RT-PCR. In evaluation of clinical prostatectomy specimens, the branched DNA assay showed a concordant detectable fusion signal in all 9 clinical samples that had fusion detected by nested RT-PCR or FISH. Moreover, branched DNA detected gene fusion in 2 of 16 prostate cancer tissue specimens that was not detected by FISH or nested RT-PCR. Conclusions: Our findings demonstrate a branched DNA assay that is effective for detection of TMPRSS2-ERG gene fusion in prostate cancer clinical specimens, thus providing an alternative method to ascertain TMPRSS2-ERG gene fusion in human prostate cancer tissue.