Laser tweezers Raman spectroscopy (LTRS) is used to study the effects of various environmental factors on staphyloxanthin biosythesis. Staphylococcus aureus cells are cultivated at the temperature ranging from 22 ℃ to 37 ℃, pH from 4.5 to 8.5, glucose mass concentration from 0 g/L to 10.0 g/L, and flavone mass concentration from 0 mg/L to 175 mg/L. The intensity of Raman band at 1523 cm-1 is used to characterize the relative content of staphyloxanthin in bacterial cells. The experimental results show that the temperature ranging from 22 ℃ to 37 ℃ has no significant effect on staphyloxanthin biosynthesis, the neutral pH is most favorable for staphyloxanthin biosynthesis, and glucose in a certain mass concentration range (0.2-5.0 g/L) promotes staphyloxanthin synthesis in a dose-dependent manner whereas flavone inhibits staphyloxanthin synthesis in a dose-dependent manner. The LTRS technology can serve as a rapid and reliable method for quantitative determination of staphyloxanthin content as well as an effective approach to screening and evaluating inhibitors of staphyloxanthin biosynthesis.