© 2014 Macmillan Publishers Limited All rights reserved. Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and as a strategy for tumor localization.