The identification of varnishes may be essential for the choice of the appropriate solvent during their removal by restorers. This recognition is obtained by UV-fluorescence emission spectroscopy with a quasi-monochromatic UV-excitation. A new portable instrument has been developed in order to implement non-destructive, contactless and in situ measurements, providing results in real time. The method is applied to the analysis of a real ancient painting. The resin-based varnish, the recipe and the state of degradation of the varnish are deduced in different locations of the painting by comparison with a database of reference varnishes. Moreover, spectral data are compared with the Fluorescence Lifetime Imaging (FLIM) analysis, performed on the same painting. Different areas containing the same varnish can then be localized on the whole painting. These results show that both UV-fluorescence methods are complementary for rapid and in situ analysis of varnishes of an entire work of art. Nevertheless, the paint layer beneath the varnish modifies the varnish fluorescence spectrum thus complicating its recognition. Indeed, the possible fluorescence of the binder of the paint layer or its reflectance spectrum must be taken into account. A systematic experimental study on fresh and aged model samples made of different varnishes, pigments and binders is presented in this work. It shows that UV-fluorescence emission spectra and diffuse reflectance spectra must be coupled to extract the fluorescence of the single varnish. Both spectra can be recorded by the presented instrument. A new theoretical approach is summarized in order to explain this phenomenon.