The human pathogenic bacterium Vibrio cholerae infects its host by expressing a protein multiplex consisting of two subunits, the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of V. cholerae and is typically detected using enzyme-linked immunosorbant assays (ELISAs). In lieu of an enzyme-linked detection method, we have developed GM1 ganglioside-functionalized fluorescent dye-encapsulating liposomes (liposomes) for the detection of CTB produced by V. cholerae in a simple microtiter plate assay. Liposomes were compared to fluorescein-labeled antibodies and enzyme-linked secondary antibodies for quantification of purified CTB. Using a criteria of the background signal plus 3xStDev, a limit of detection of CTB for the liposomes was 0.34 ng/mL, which was comparable to the ELISA, but significantly lower than the fluorescein-labeled anti-CTB antibody used for the same purpose. The signal enhancement provided by the liposomes was substantial when compared to the fluorescein labeled antibody. For example, using a criteria of signal to noise of 1.5:1, a limit of detection of CTB for the liposomes was 0.17 ng/mL, which was 178 times lower than the fluorescein-labeled anti-CTB antibody. In addition, the liposomes required shorter assay times and less variability than ELISA. Liposomes were optimized with respect to phospholipid and ganglioside concentrations and used to probe culture supernatants from V. cholerae El Tor C6706 grown in DMEM, LB, and AKI media for the presence of CTB. The cost of using liposomes was calculated to be a fraction of either ELISA or fluorescein-labeled antibodies.