Tuberculosis remains a major global health concern, especially in the developing world, and monitoring/early detection of the disease relies on low cost technologies that provide rapid and accurate results. Mycobacterium tuberculosis is the responsible bacterial pathogen and it is currently estimated by the World Health Organisation (WHO), that one quarter of the world's population, mainly in the developing world, is infected with TB. The overall aim of this work was to advance a screening electrochemical sensor for label free detection of Ag85B, a member of the Antigen 85 complex—major secretary protein of M. tuberculosis and biomarker for disease. An indirect ELISA Ag85B assay was optimised with capture antibody and antigen levels determined via a checkerboard titration (0.625 μg ml−1 and 2.5 μg ml−1 respectively). Following assay development, crosslinking of the bioreceptor Anti-Ag85B onto electrochemically deposited gold nanoparticle (AuNP) modified carbon electrodes was achieved and Ag85B binding successfully evaluated electrochemically via cyclic voltammetry. Following each modification step, ΔEp of a redox probe was monitored and overall results show that GCE/AuNP/anti-Ag85B electrochemical transducers are a viable method for Ag85B detection, capable of measuring antigen levels <2.5 μg ml−1.