A plethora of semiconductor quantum dot (QD)-based probes that rely on Forster resonance energy transfer (FRET) have been developed for the optical detection of a wide array of biological targets. To date, the vast majority of these probes have utilized one-step energy transfer between individual donor-acceptor pairs. Here, we report a new multidonor concentric FRET configuration that comprised two fluorescent dyes assembled around a central CdSeS/ZnS QD through peptide linkers. One of these dyes, either Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647), served as an acceptor for both the central QD and the other coassembled dye, Alexa Fluor 488 (A488). The unresolved emission between the A488 and the QD precluded a standard analysis of FRET efficiency from quenching of donor emission intensity or decay time, instead necessitating an analysis of the two energy transfer pathways from deconvolved excitation spectra. When A647 was the terminal acceptor, both the QD-to-A647 and A488-to-A647 energy transfer pathways could be interrogated with blue light, but only the former could be interrogated with violet light. The different degrees of A647 sensitization between these two excitation wavelengths was a predictable function of the above energy transfer efficiencies and dye stoichiometry, and was exploited for quantitative bioanalysis through an excitation ratio, which is in contrast to the conventional use of an emission ratio with FRET-based probes. Detection of the activity of nanomolar concentrations of trypsin, a model protease that hydrolyzed the A488-labeled peptide linker, was demonstrated using both a fluorescence plate reader and a low-cost, compact device that used two low-power light-emitting diodes (LEDs) as excitation sources and a silicon photodiode to detect A647 emission. This multidonor concentric FRET configuration represents a new modality for ratiometric biosensing with QDs and is potentially useful for portable in vitro diagnostics.