Invasive aquatic species introductions cause tremendous environmental and economic damage. Conservation and management efforts will benefit from rapid, inexpensive, and accurate on-site methods to detect harmful aquatic species to prevent their introduction and spread. Here, two technologies, environmental DNA (eDNA) sampling and Light Transmission Spectroscopy (LTS), were combined to address this need. Specifically, eDNA filtering and extraction methods were used to isolate DNA from: (1) lake water samples that were seeded with a microscopic fragment of five high-risk invasive species and (2) untreated samples from lakes infested with the invasive zebra mussel, Dreissena polymorpha, followed by polymerase chain reaction (PCR) amplification. LTS was then used to detect size shifts resulting from hybridization of PCR products with nanobeads covered with species-specific oligonucleotide probes. The results demonstrate that coupling eDNA sampling with LTS species detection can provide a sensitive and real-time solution for screening real-world water samples for invasive species.