Conditional stability constants and binding capacities are important parameters with which to estimate the biological availability of metal ions in aqueous solution in the presence of dissolved natural organic matter (fulvic acid, organic matter in natural waters or in aqueous extracts of forest litter). Determination of these parameters depends on analytical methods that can distinguish between free and organically bound metal ions. This speciation is difficult, mainly because natural organic matter typically is a complex mixture. In this paper, multi-wavelength molecular fluorescence spectrometry is evaluated prototypically as a method for the determination of stability constants and binding capacities for Cu(II) and Al(III) complexation by dissolved organic matter in a juniper leaf litter extract. Equilibrium ion exchange quantitation and electron spin resonance spectroscopy served as quantitative and qualitative reference methods, respectively. Three types of binding site for Cu and Al could be differentiated qualitatively by the reaction patterns of various wavelength regions of the total luminescence spectrum of the leaf litter extract in response to increasing metal ion addition. For both Cu (pH 6) and Al (pH 5), binding parameters for the two types of binding site forming the most stable complexes were deduced self-consistently from reactions evaluated at selected excitation/emission wavelength pairs.