I worked on this 30 years ago. First of all, you need to be certain that you are working with a pure compound, not a mixture of compounds. You need to isolate it by some kind of techniques, e.g., TLC, HPLC, wet chemistry, and so on. Once you know that you have a single compound, find the UV absorption spectra using a spectrophotometer. The lambda max is the excitation wavelength. Then put the solution into the flurormeter, set the excitation wavelength and scan for emission wavelength to find the maximum intensity.

The previous answer is right. You need to have the pure substance, because the presence of another one might cause interference as the peaks might overlap.

Thank You.