A new rapid, specific and sensitive method for assay of recombinant CFP10-ESAT6 amalgamation proteins from Mycobacterium tuberculosis was proposed. The method used streptavidincoated magnetic beads to enrich the specific biotinylated anti-CFP10 antibody, then adopted a sandwich-type enzyme linked immunosorbent assay technology with two kinds of monoclonal antibodies: biotinylated anti-CFP10 antibody and HRP-labeled anti-CFP10 antibody to identify the target CFP10-ESAT6 proteins, and finally detected chemiluminescence intensity by a small home-made optical sensor. It was shown that, the corresponding chemiluminescence intensity had a good logarithmic linear response to the concentration of CFP10-ESAT6 proteins when ranging at 1 - 1000 ng/mL, and the correlation coefficient is 0.9937. The proposed method could detect the CFP10-ESAT6 proteins with low detection limit (1 ng/mL) and the detection time could be controlled within 45 min. Compared with commonly used detection methods of M. tuberculosis, this method was easy to operate, faster, and of higher sensitivity. The achievement of the quantitative detection of CFP10-ESAT6 proteins has important scientific significance and wide application prospects in tuberculosis control.