In this work, we describe a new “signal-on” electrochemical biosensor based on a competitive immunoassay with a label-free format for the facile, sensitive and cost-effective determination of oxytetracycline (OTC). The device pattern was designed and printed on low-cost filter paper to construct a disposable paper-based analytical device (PAD) using a wax printing technique. The sensor consisted of a capture anti-OTC antibody (anti-OTC) and OTC-conjugated bovine serum albumin (OTC-BSA). Anti-OTC was first anchored onto the functionalized PAD, followed by the competitive binding of the OTC target and OTC-BSA. The amount of the captured OTC was examined by observing the signal response of the redox [Fe(CN)₆]^3−/4−. In the presence of OTC, the current response was significantly increased with increasing OTC concentration, whereas the signal response was negligible in the absence thereof. The detected OTC concentration was linear over a range of 1−200 ng mL^-1, and the corresponding values of 0.33 and 1.1 ng mL^-1 were obtained for the limit of detection (LOD) and limit of quantitation (LOQ), respectively. This introduced sensor displayed high sensitivity and specificity, and the amounts of OTC were successfully quantified in agricultural products. This developed signal-on sensor may be an alternative tool for determining OTC residues and broader antibiotic targets.