The total bacterial count detection reagent uses enzyme substrate technology to detect the total bacterial count in water. The reagent contains a variety of unique enzyme substrates, each designed for different bacterial enzymes. When different enzyme substrates are decomposed by enzymes released by different bacteria, they release fluorescent groups. By observing the number of fluorescent cells under the ultraviolet lamp with the wavelength of 365 nm or 366 nm, the total value of colonies can be obtained by looking up the table.