At every stage of the vaccine’s development, including research and development together with the routine production (in-process control of batch release), quality control testing is a crucial operation for ensuring the efficacy, safety and consistency of final products. Conventional quality control tests rely on a combination of biochemical, physicochemical, and cell-based assays. In this study, we report a quality control test based on an electrochemical aptamer-based sensor with a specific application to Diphtheria vaccine. First, specific aptamers for Diphtheria are successfully selected and characterised by using SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. A total of 11 rounds were performed to select five sequences with variable affinities to Diphtheria toxoid. The aptamer D1 exhibiting the best affinity towards the Diphtheria toxoid (Kd= 1.4 nM) was employed to develop an electrochemical aptasensing platform. The selected aptamer was conjugated to a thiol group to enable its immobilization on screen-printed gold electrodes (SPGEs). Then, the determination of Diphtheria toxoid was performed in a label-free mode by monitoring the electrochemical signal before and after the formation of the aptamer-target complex. Excellent analytical performance was obtained within a wide linear range (0.008 to 290 ng/mL) with the low detection limit of 1.92 ng/mL. Given the combined use of Diphtheria with Pertussis and Tetanus, cross-reactivity tests were centred on these two vaccines. The aptasensor showed high selectivity and specificity for Diphtheria compared to a negligible response for the non-specific toxoids. Furthermore, the applicability of the aptasensor was successfully investigated for the detection of Diphtheria toxoid in real vaccine samples. The developed aptasensor shows a great promise as a fast, cost-effective and accurate method for vaccine quality control testing.