Objective To establish a near‐infrared fluorescent dye‐immunomagnetic beads coupling method for quantitative de‐tection of Mycobacterium tuberculosis early secretory antigenic target‐6(ESAT‐6) .Methods Near‐infrared fluorescent dye ,dylight 800 ,was used to mark ESTA‐6‐targeting monoclonal antibodies ,and the surface of nano‐magnetic beads were coated with ESAT‐6‐targeting polyclonal antibodies .Double antibody sandwich method was used for magnetic separation of conjugates and dissociants . Portable high‐sensitivity and low‐noise excitation fluorescence detector was used to detect the intensity of magnetic combination ,so as to test the ESAT‐6 content in test samples .Results The detecting linear range of this method was 2 .4-750 .0 ng/mL ,and the minimum detection limit was 0 .48 ng/mL .The recovery rate was 96% at a concentration of 10 ng/mL ,and at a concentration of 50 ng/mL the recovery rate was 95% .The between‐run coefficient of variation(CV)value was 5 .8% ,and the within‐run CV value was 4 .3% .The specificity and sensitivity of this method for detecting ESAT‐6 in clinical pleural effusion samples were 80% and 95% , respectively .Conclusion This method might have wide linear range ,high sensitivity and good stability in the detection of ESAT‐6 .