The photophysics of a europium(III) complex of 1,4,7,10-tetraazacycododecane-1,4,7-triacetic acid-10-(2-methylene)-1-azathioxanthone was investigated in three buffer systems and at three pH values. The buffers—phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and universal buffer (UB)—had no effect on the europium luminescence, but a lower overall emission intensity was determined in HEPES. It was found that this was due to quenching of the 1-azathioxanthone first excited singlet state by HEPES. The effect of pH on the photophysics of the complex was found to be minimal, and protonation of the pyridine nitrogen was found to be irrelevant. Even so, pH was shown to change the intensity ratio between 1-azathioxanthone fluorescence and europium luminescence. It was concluded that the full photophysics of a potential molecular probe should be investigated to achieve the best possible results in any application.