Fluorescent tracers are widely used for assessment of spray quantity in the field due to their relatively high sensitivity, low cost and user safety. However, many concerns have been raised over their measurement accuracy due to questions of stability of fluorescence during tests. Stable analysis of fluorescence is essential to ensure accurate evaluation of pesticide spray application efficiency. The objective of this research was to determine the stability of fluorescent intensity of five tracers dissolved in solutions with various pH conditions in an effort to minimize analytical errors in the measurement of spray deposition and drift. The fluorescent intensity of five fluorescent tracers commonly used for the quantitative assessment of spray deposition and off-target loss was investigated with wash solutions over pH conditions from 6.9–10.4. The tracers selected in the tests were Brilliant Sulfaflavine (BSF), Fluorescein, Pyranine, Tinopal, and Eosin. The fluorescence of Pyranine was the most sensitive to the solution pH conditions, followed by Fluorescein and Tinopal, while BSF and Eosin had a nearly constant fluorescent intensity over the pH range from 6.9–10.4. The fluorescence of Fluorescein increased 1.3 times, Tinopal 1.25 times, and Pyranine 3.0 times as the pH value increased from 6.9–8.4, but it became nearly constant when pH value was greater than 8.4. However, Pyranine, Fluorescein, and Tinopal showed much stronger fluorescence than BSF and Eosin. A solution containing Fluorescein at pH 8.4 and higher demonstrated 83 times greater fluorescent intensity than the solution containing the same amount of BSF. In conclusion, the fluorescence of tracers should be examined under various pH conditions during the selection of tracers for pesticide spray deposition and drift trials.