A hepatitis c virus antigen-antibody joint detection reagent box is characterized by comprising a calibrator(1), a double-marker enzyme conjugate (2), a negative and positive contrast (3), light-emitting liquid (4) and a micropore coated plate (5), wherein the light-emitting liquid contains light-emitting liquid 1 and light-emitting liquid 2, the light-emitting liquid 1 contains luminal 0.7 g / L, cinnamic acid 0.9 g / L, 4-iodophenylboronic acid 0.2 g / L, iodobiphenol 0.25 g / L, dimethylformamide 25 ml / L, polyving akohol 5 g / L, polyvinylpyrrolidone 8 g / L, polyethylene glycol 600 3 g / L, ethylenediamine tetraacetic acid 4 g / L, gentamicin sulfate 1600 thousands / L, urea peroxide 0.4 g / L, and pH 9.0 Tris buffer solution 0.1 mol / L. The light-emitting liquid 2 contains acridinium ester derivative 0.1 mg / ml, polyethylene glycol 600 3 g / L and 0.1 mol / L of pH 9.0 Tris buffer solution containing 0.1% of TWEEN-20. The invention further discloses a preparation method and a using method of the reagent box. The hepatitis c virus antigen-antibody joint detection reagent box has the advantages of being quick in reaction and low in cost.