The attractive photophysical properties of luminescent nanoparticles have prompted numerous studies on their synthesis and biological applications. Due to the complexity of components, sizes, morphologies, and strong light scattering of the nanoparticles the absorption spectrum alone is not enough for characterising and analysing the real absorption band of a nanoparticle product. Instead, the excitation spectrum is necessary for this purpose. To address the importance of the excitation spectrum in the utilisation of the photophysical properties of nanoparticles for cellular imaging, we compared the absorption and excitation spectra of a small organic molecule (rhodamine 6G) and commercially available nanoparticles (semiconductor quantum dots). Rhodamine 6G shows a good superimposition between its absorption and excitation spectra. However, the quantum dots do not, due to the presence of impurities and the polydispersity in nanoparticle size and shape. We demonstrated, by either spectrometer or cellular imaging, that excitation of the quantum dots at the maximum absorption peak will not result in the highest emission intensity. Instead, the best excitation wavelength for the nanoparticles can be revealed by the excitation spectrum. This has usually been overlooked by chemists and biologists.