The influence of photocatalytic degradation on molecular size fractionation (0.45 μm filtered, 100 kDa, 30 kDa and 3 kDa) of humic acid as a model compound of natural organic matter was investigated. The results were evaluated using UV-vis parameters, dissolved organic carbon (DOC) and excitation emission matrix (EEM) fluorescence spectral features. EEM fluorescence signatures displayed an irradiation period dependent transformation of humic-like fluorophores to fulvic-like fluorophores in accordance with the photocatalytic mineralization of HA. Molecular size distribution profiles expressed the formation of lower molecular size (<3 kDa) fractions through oxidative degradation of humic acid of higher molecular size fractions (100 kDa and 30 kDa fractions). The fluorescence-derived index (fluorescence intensity (FI), represented by the ratio of the emission intensity at λemis = 450 nm to that at λemis = 500 nm, following the excitation at λexc = 370 nm) was also investigated. The use of EEM features has proven to be a useful tool for monitoring the effect of photocatalytic degradation on the structure and molecular size distribution profile of HA.