Dengue, or break-bone fever, is the most common mosquito-borne viral disease of humans with over half the world's population at risk for infection. Dengue has a wide spectrum of clinical manifestations, from self-limited febrile illness to fatal hypovolemic shock, and because of this, dengue is difficult to distinguish from many other infections based on clinical characteristics alone. Diagnostic testing is therefore critical to accurately identify dengue virus (DENV)-infected patients and also rule out dengue in patients with undifferentiated fever. Unfortunately, current diagnostics for early DENV detection consist of point-of-care or laboratory-based antigen tests that lack sensitivity or molecular assays that are laborious to perform or lack the test characteristics necessary for routine use. To address these limitations, we developed a single-reaction, multiplex, real-time RT-PCR for the detection, quantitation, and serotyping of dengue viruses from patient serum or plasma. We demonstrate that this diagnostic test is more analytically sensitive than a commonly used reference molecular assay, and is able to detect viral RNA and provide serotype information in specimens collected more than 5 days after fever onset and from patients who had already developed anti-DENV IgM antibodies. This unique combination of sensitivity and serotyping capability in a simple, single-reaction format represents a step forward in dengue diagnostics.