We demonstrated the feasibility of using ozonation to enhance the performance of dsDNA binding dye SYBR Green I in the fluorescence measurement of total bacterial load in water. Unlike its membrane permeable but expensive equivalent such as SYTO82 dye, SYBR Green I is many times cheaper but membrane impermeable. Ozonation allowed SYBR Green I dye to permeate the membrane and bind with the dsDNA within by first breaching it. Using E. coli K12 bacteria at serial dilution ratios from 1/1 (980 CFU mL-1) to 1/200, we achieved corresponding quantification from 618.7 ± 9.4 to 68.0 ± 1.9 RFU (100 to 11.00% normalized RFU). In comparison, plate counting and optical density measurement were only able to quantify up till a serial dilution ratio of 1/50 (40 CFU mL-1 and 0.0421, respectively). Most importantly with ozonation, the sensitivity of SYBR Green I dye based fluorescence measurement was improved by ∼140 to 210% as compared to that without ozonation. Given its low electrical power consumption, lab-on-chip compatibility and reagent-less nature, ozonation is highly compatible with portable fluorimeters to realize low-cost monitoring of total bacterial load in water.