The invention relates to a method for detecting the total number of bacteria based on the fluorescent method of NADH, in particular to a novel method for rapidly detecting the total number of colon bacteria based on the fluorescent characteristic of the NADH in a colon bacteria cell and the stable-content characteristic of the NADH in the colon bacteria cell, which comprises the processes of extracting NADH in the colon bacteria cell, detecting the content of the NADH in the colon bacteria cell, detecting the fluorescent intensity of the NADH in the colon bacteria cell with a fluorescent spectrophotometer and rapidly detecting the total number of bacteria. The fluorescent detection limit of the NADH is 1 nM, and the content of the NADH ranges from 10 nM to 80 microns and keeps a favorable liner relationship (R = 0.9905) with the fluorescent intensity. The colon bacterial cell is obtained in a decentering way, the NADH in the colon bacteria cell is extracted in a Tris-HCl method, the fluorescent intensity of extracting solution of the NADH is extracted with the excitation wavelength of 342 nm and the emission wavelength of 461 nm, and the colon bacteria of 10 cfu/mL can be detected in one hour. The method adopts novel detection theory, has rapid detection speed and good repeatability, is sensitive and easy, is suitable for quantitatively detecting the number of colon bacteria in the fields of food sanitation and safety, environment detection, and the like, and can replace the traditional method for detecting the number of living cells.