Whole-cell biosensors utilize the natural processes of living cells to detect changes in their environment. The Pseudomonas putida strain TVA8 bacterial-based biosensor is used to sense parts-per-billion levels of chemicals or inducers (i.e. trichloroethylene-TCE) through bioluminescence. However, the technical challenge in detecting changes in inducer concentrations is to engineer a quantitative link between the bioluminescence signal and the inducer concentration. Because whole cells of bacteria are used, there is extreme sensitivity to environmental perturbations such as temperature. In order for a sensor to “self-calibrate,” the quantitative effect of variations in environmental conditions must be determined. This study focuses on the influence of temperature, cell number and inducer concentration on the bioluminescence signal and growth of P. putida strain TVA8. Cells were grown in a shaking liquid culture, exposed to the target analyte (TCE) to induce bioluminescence, and allowed to grow into logarithmic growth phase. The bioluminescence signal and cell growth were measured with a Zylux FB14 luminometer and a Spectronic 21D spectrophotometer, respectively. Optimal temperatures for bioluminescence (23 °C) and growth rate (34 °C) were determined, as well as a method of “resetting” the bioluminescence signal for sensor calibration. This inhibition of signal was a result of a sudden increase of temperature from 25 °C to 37 °C. The bioluminescence signal was found to decrease rapidly, with an average of 95.388 ± 1.59% and 99.858 ± 0.093% of the maximum bioluminescence eliminated after 2 and 15 minutes, respectively.