The invention discloses a method for determining the content of glutathione in bacterial cells. The method comprises the following steps: drafting a standard curve of different concentrations of glutathione to the fluorescence intensity; adopting a plate colony-counting method to carry out colony counting; setting a bacterial liquid experiment group for determination and a background fluorescence control group; processing the experiment group and the control group, and detecting by adopting a fluorescence intensity detection method; calculating according to the standard curve to obtain the glutathione content, and calculating according to flat counting to obtain the quantity of sample bacteria; and calculating a ratio of the glutathione content to the quantity of the corresponding bacteria to obtain the content of glutathione in a certain quantity of bacterial cells. An mBBr reagent is adopted in the invention, can go through the cell walls of Gram-positive bacteria, enters the cells, and combines with glutathione to generate a stable fluorescence derivative, and the bacteria are cracked by acetonitrile, so a problem of the wall breaking of the Gram-positive bacteria of the measurement method is solved.