The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. The 6-kDa early secreted antigenic target (ESAT6) and 10-kDa culture filtrate protein (CFP10) are secreted early by virulent Mycobacterium tuberculosis (M. tb) and are not present in the non-virulent Bacillus Calmette–Guerin (BCG). In this study, we used a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique to screen for a functional ssDNA aptamer “antibody” that specifically bound to ESAT6-CFP10 (CE) protein. The selected single ssDNA aptamers (CE24 and CE15) demonstrated the highest specificity and binding affinity to CFP10 (CE24: K_d = 3.75 × 10⁻⁷ M) and ESAT6 (CE15: K_d = 1.6 × 10⁻⁷ M). We further detected CFP10 and ESAT6 proteins in serum samples from active pulmonary tuberculosis (TB) patients, extrapulmonary TB patients and healthy donors by using an enzyme-linked oligonucleotide assay (ELONA). The results showed that the sensitivity and specificity were 100% and 94.1% (using CE24 aptamer-based ELONA) and 89.6% and 94.1% (using CE15 aptamer-based ELONA), respectively. A good correlation was observed between aptamer-based ELONA and T-SPOT TB assay. Thus, our study suggests that CE24 and CE15 have potentially broad applications as early antigen diagnostic agents not only for active pulmonary TB, extrapulmonary TB, but also possibly for latent TB infection and TB with immune-deficiency.