Aims: The aim of this study was to develop and optimize a system for the detection of multiple biological targets in a single sample based on enzymatic bionanotransduction. Method and Results: We used biological recognition elements (antibodies, DNA sequences) linked to DNA templates with T7 promoter regions for detection of specific target molecules. In vitro transcription of DNA templates bound to target molecules produced RNA nanosignals specific for every target in the sample. An enzyme-linked oligonucleotide fluorescence assay (ELOFA) provided a correlation between nanosignal profiles and target concentrations. The system was capable of detecting and distinguishing three species of specific immunoglobulin G (IgG) molecules at a level of 0.2 ng, mixed protein and DNA targets and single sample detection of Escherichia coli O157 micro-organisms and Staphylococcal enterotoxin B (SEB). Conclusions: This report provided proof of concept for the use of enzymatic bionanotransduction with multianalyte biological detection based on differential nanosignal hybridization along with the application of this system to pathogen/toxin detection. Significance and Impact of the Study: This system has the potential to be used as a tool for detection of multiple foodborne and environmental pathogens, toxins and targets of interest in a single sample.