This paper Used laser light sources and xenon lamp light source of online fluorescence spectrometry to determine the optical content of rhodamine B, vitamin B2, fluorescein and fluorescein isothiocyanate, and compared the light intensity of laser light sources and xenon lamp light source. Methods:The optical concentration of Rhodamine B, vitamin B2, fluorescein and different hydrogen sulfate fluorescein were equally 10 μg·mL-1; Integration time was 100 ms, and the average value was measured by three times detection. Maximum absorption wavelength determinated by online fluorescence spectrometry were respectively 580, 450, 488 and 510 nm; The maximum emission wavelength were respectively 594, 530, 525 and 524 nm. The maximum absorption wavelength detemineted by UV spectrophotometry were respectively 557, 441, 481 and 490 nm; Maximum emission wavelength detected by fluorescence method were respectively 586, 520, 519, 520 nm. By measuring medicine, we have found that the laser light source of the fluorescent light intensity is stronger than the xenon lamp light source of the fluorescent light intensity. The reason is not only related to the light source, and with the size of drug molecules conjugated system, large conjugated PI coplanar and rigid degree, the kinds of molecular substituents on the matrix, molecules of the external environment such as temperature, solvent, acid and alkali solution, such factors as the excitation light also can affect the fluorescent efficiency. Laser light sources and xenon lamp light source of the fluorescent light intensity size is rhodamine B> vitamin B2> fluorescein> fluorescein isothiocyanate in order. Laser light source online fluorescence spectrometry , to a certain extent, fill in the blank of online fluorescence spectrometer application in food, medicine, trace detection.