Electrochemical aptamer-based (E-AB) biosensors suffer from sensor-to-sensor signal variations due to the variation of the total number and the heterogeneity of probes immobilized on the electrode surface, with the former attracting more attention. As such, a calibration process to correct for such variations is required for this type of sensor, causing inconvenience and inaccessibility in harsh sensing environments such as blood samples, which has dramatically limited the widespread clinical use of biosensors. In response, here, we have adopted E-AB sensors to achieve calibration-free measurements of small biological/drug molecules. Specifically, we employ one probe-attached redox reporter and a second intercalated redox reporter to generate two signals, achieving good sensor-to-sensor reproducibility and thus obviating the need for calibration. We first demonstrated the capability of E-AB sensors for the accurate measurement of kanamycin, tobramycin, and adenosine triphosphate (ATP) in phosphate-buffered saline (PBS) buffer, achieving concentration ranges of approximately 4.7 × 103-, 2.0 × 103-, and 12.7-fold, respectively. Then, we applied this calibration-free approach to the measurement of these three target molecules directly in undiluted serum, achieving a concentration precision of a few micromolars.