Flow cytometry and spectrophotometry were used to measure the cell numbers and absorbances of three common cultures including Escherichia coli S17-1, Synechocystis sp. PCC 6803 and Bacillius subtilis 168 at different concentrations in the laboratory. Then the absorbance values and cell numbers of each culture were compared. The function relationship between absorbance and cell numbers was established. The results showed that the absorbance value and cell numbers of the three common cultures in the laboratory were significantly positively correlated with the concentration at the certain concentration. It was verified that the absorbance value of the culture can reflect the number of cells during the culture process using the fitting function. The relationship between the absorbance and cell number of three strains including Escherichia coli S17-1, Synechocystis sp. PCC 6803 and Bacillius subtilis 168 was established. It will provide the foundation for developing biological experiments to determine the cell number and downstream molecules in the culture using a spectrophotometer. 