There is an increasing desire to measure multiple analytes simultaneously for disease management and detection. However, in the case of invasive devices, it would be better to obtain one small sample and immediately be able to detect the analytes rapidly, as in the case of self-monitoring blood glucose, without the need for additional steps, arrays, or reagents. Electrochemical impedance spectroscopy is used to measure the interaction between ultralow levels of analyte and molecular recognition element in a label-free and rapid manner. Gold nanoparticles were attached to antibodies against interleukin-12 and tumor necrosis factor-α, typical inflammatory markers found with near overlapping responses, on an impedance spectroscopy based biosensor. Cross-reactivity and specificity of tuned antibodies were verified using ELISA. Impedance frequency was quantified by concentration gradients of marker against the device. The natural impedance frequency for interleukin-12 (5.00 Hz) was tuned to a lower frequency four Hertz away from one another for better signal processing. This was accomplished without significantly altering the lower limits of detection (<4 pg ml−1 and ∼60 pg ml−1 for interleukin-12 and tumor necrosis factor-α, respectively), no cross-reactivity and specificity as determined by ELISAs. With modeling the nanoscale effects and further development, a larger tuning will be possible for making a better multiplexed sensor. Although interleukin-12 and TNF-α equivalent circuit calculations were modeled here, a sensor with the potential to measure multiple markers at once might offer a solution on the sensor front for simplified management of conditions such as diabetes, where both glucose and hemoglobin A1c values could be obtained.