The genetic material (DNA fragments of cellular or extracellular origin) that organisms leave behind in nonliving components of the environment such as water, soil, or sediments is defined as environmental DNA (eDNA). Recently, the use of eDNA has been recognized as an effective method for aquatic invasive species early detection and surveillance. We developed molecular markers for eDNA detection of the African jewelfish (Hemichromis letourneuxi) in and around Loxahatchee National Wildlife Refuge, Florida. The lower limit of detection using these markers was determined as well as the effect of fish density and time on detection using controlled experiments. Specificity and sensitivity of these markers was tested in aquarium trials and also in field samples. Results showed that developed markers (probe and primers) for Taqman assays were sensitive and specific for eDNA detection via traditional and quantitative PCR methods (qPCR). The observed theoretical minimal qPCR detection level, based on standard curve analysis for this species, was approximately 0.0002 ng/uL (R2 = 0.90) at a PCR cycling threshold (CT) of 28.5–29. There was a positive and significant relationship between fish density and eDNA detection with detection probabilities ranging from 0.32–1.00 depending on fish density. A negative and significant relationship between average CT values and density further corroborated our findings that target eDNA increased with increasing fish density. Developed markers detected the presence of H. letourneuxi in the canal adjacent to but not in Loxahatchee National Wildlife Refuge. The single positive found in the canal adjacent to Loxahatchee National Wildlife Refuge showed a similar CT value to the observed average for density of three fish per aquarium.