A rapid method for readily detecting the total numbers of viable bacterial cells in numerous samples (including surface water, solid inoculants, and soil samples) is reported using a newly developed hand-held fluorometer and a fluorescent dye Calcein UltraGreen™ AM. Compared to the traditional plate counting method that requires 48 hours of cultivation, the newly established method does not require any incubation time, making the detection method faster and more convenient. The portable rapid detection fluorometer has a wide dynamic range of relative fluorescence intensity from 45 to 30 133. It can detect bacterial concentration ranging from 105 to 1010 cells per mL. This newly established method has good applicability for accurately and quickly detecting the cell number of viable bacteria in various samples. The results of the fluorescence-based method were compared with those of the traditional plate counting method, and it was found that the relative standard deviation was less than 6%. This new rapid measurement system provides a robust method for the rapid on-site detection of viable bacteria.