A molecular diagnostic method for robust detection of Ebola virus (EBOV) at the point of care (POC) directly from blood samples is described. This assay is based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) of the glycoprotein gene of EBOV. Complete reaction formulations were lyophilized in 0.2-mL polymerase chain reaction tubes. RT-LAMP reactions were performed on a battery-operated isothermal instrument. Limit of detection of this RT-LAMP assay was 2.8 × 102 plaque-forming units (PFU)/test and 1 × 103 PFU/test within 40 minutes for EBOV-Kikwit and EBOV-Makona, respectively. This assay was found to be specific for the detection of EBOV, as no nonspecific amplification was detected in blood samples spiked with closely related viruses and other pathogens. These results showed that this diagnostic test can be used at the point of care for rapid and specific detection of EBOV directly from blood with high sensitivity within 40 minutes.