Nowadays phototherapy is widely used for treatment of various diseases. However, efficient application of phototherapy requires an understanding of light interactions with main endogenous chromophores (e.g., hemoglobin, bilirubin, and water) in tissue. In particular, bilirubin is the target chromophore in the treatment of neonatal jaundice, which is the most common disease affecting up to 80% of preterm infants. The most frequently recommended treatment technique for this disease is phototherapy with blue light in combination with conventional drug therapy. To follow threshold total serum bilirubin (TSB) concentration guidelines, it is essential to estimate TSB concentration accurately. The gold standard biochemical analysis is invasive and bulky. Moreover, noninvasive methods do not provide sufficient reproducibility and accuracy. In this research, the fluorescence sensing of bilirubin with human serum albumin complexes was studied. The fluorescence time course during light irradiation (central wavelength: 467 nm and power density: 12.13 mW cm-2) was demonstrated to depend on the initial concentration. Specifically, for the bilirubin concentration C = 18.65 μM, an insignificant fluorescence signal increase was observed during the first 30 minutes of light irradiation, while for bilirubin concentration C = 373 μM, the fluorescence signal did not reach maximum during 2.5 hours of light irradiation. Thus, fluorescence sensing might show increased accuracy when used with other noninvasive bilirubin sensing methods.