Measuring the absolute protein expression quantity for a specific promoter is necessary in the fields of both molecular biology and synthetic biology. The strength of a promoter is traditionally characterized by measuring the fluorescent intensity of the fluorescent protein downstream of the promoter. Until now, measurement of the absolute protein expression quantity for a promoter, however, has been unsuccessful in synthetic biology. The fact that the protein coding sequence influences the expression level for different proteins, and the inconvenience of measuring the absolute protein expression level, present a challenge to absolute quantitative measurement. Here, we introduce a new method that combines the insulator RiboJ with the standard fluorescence curve in order to measure the absolute protein expression quantity quickly; this method has been validated by modeling verification. Using this method, we successfully measured nine constitutive promoters in the Anderson promoter family. Our method provides data with higher accuracy for pathway design and is a straightforward way to standardize the strength of different promoters.