Bromothymol blue may be used for observing photosynthetic activities, or as a respiratory indicator (turns yellow as CO2 is added). A common demonstration of BTB's pH indicator properties involves exhaling through a tube into a neutral solution of BTB. As carbon dioxide is absorbed from the breath into the solution, forming carbonic acid, the solution changes color from green to yellow. Thus, BTB is commonly used in science classes to demonstrate that the more that muscles are used, the greater the CO2 output.

Bromothymol blue has been used in conjunction with phenol red to monitor the fungal asparaginase enzyme activity with phenol red turning pink and bromothymol blue turning blue indicating an increase in pH and therefore enzyme activity. However, a recent study suggests that methyl red is more useful in determining activity due to the bright yellow ring form in the zone of enzyme activity.

It may also be used in the laboratory as a biological slide stain. At this point, it is already blue, and a few drops are used on a water slide. The cover slip is placed on top of the water droplet and the specimen in it, with the blue coloring mixed in. It is sometimes used to define cell walls or nuclei under the microscope.

Bromothymol is used in obstetrics for detecting premature rupture of membranes. Amniotic fluid typically has a pH > 7.2, bromothymol will therefore turn blue when brought in contact with fluid leaking from the amnion. As vaginal pH normally is acidic, the blue color indicates the presence of amniotic fluid. The test may be false-positive in the presence of other alkaline substances such as blood or semen, or in the presence of bacterial vaginosis.