A method for quantitatively co-expressing multiple proteins in vitro, comprising the steps of: establishing a standard curve: establishing standard curve of protein concentration-luminescence intensity relationship for each co-expressed protein using corresponding standard protein; creating separate vectors containing each target protein gene for expressing each target protein respectively; establishing an equation of quantitative relationship between concentration percentage of each of target proteins and concentration percentage of the corresponding vector; wherein the separate vectors in Step containing the target protein genes are added at different concentration ratios to in vitro cell-free protein synthesis system for protein synthesis reaction; after a specified reaction time, a luminescence value for each target protein in the reaction solution is obtained; concentration of each target protein product is calculated according to the standard curve shown in Step, and an equation of quantitative relationship between concentration percentage of each target protein product and concentration percentage of the corresponding vector is obtained by fitting; in in vitro cell-free protein synthesis system, the total concentration of the vectors remains the same; calculating concentration and concentration ratio of the vectors for quantitatively co-expressing multiple proteins; wherein, according to target concentration ratio relationship of the multiple target proteins to be expressed, the concentration and concentration ratio of the vector for each of the multiple target proteins to be expressed are calculated by using the equation established in Step; quantitatively co-expressing the multiple proteins; wherein, according to the required concentration or concentration ratio of each target protein vector obtained in Step, corresponding amount of the independent vector of each target protein is added to the in vitro cell-free protein synthesis system as described in Step, and after the specific period of time defined in Step, the co-expressed multiple target proteins are obtained; wherein the multiple target proteins are each independently luminescent protein or fusion protein carrying a luminescent label. establishing a standard curve: establishing standard curve of protein concentration-luminescence intensity relationship for each co-expressed protein using corresponding standard protein; creating separate vectors containing each target protein gene for expressing each target protein respectively; establishing an equation of quantitative relationship between concentration percentage of each of target proteins and concentration percentage of the corresponding vector; wherein the separate vectors in Step containing the target protein genes are added at different concentration ratios to in vitro cell-free protein synthesis system for protein synthesis reaction; after a specified reaction time, a luminescence value for each target protein in the reaction solution is obtained; concentration of each target protein product is calculated according to the standard curve shown in Step, and an equation of quantitative relationship between concentration percentage of each target protein product and concentration percentage of the corresponding vector is obtained by fitting; in in vitro cell-free protein synthesis system, the total concentration of the vectors remains the same; calculating concentration and concentration ratio of the vectors for quantitatively co-expressing multiple proteins; wherein, according to target concentration ratio relationship of the multiple target proteins to be expressed, the concentration and concentration ratio of the vector for each of the multiple target proteins to be expressed are calculated by using the equation established in Step; quantitatively co-expressing the multiple proteins; wherein, according to the required concentration or concentration ratio of each target protein vector obtained in Step, corresponding amount of the independent vector of each target protein is added to the in vitro cell-free protein synthesis system as described in Step, and after the specific period of time defined in Step, the co-expressed multiple target proteins are obtained; wherein the multiple target proteins are each independently luminescent protein or fusion protein carrying a luminescent label.