We investigated the bioavailability characteristics of chromophoric dissolved organic matter (CDOM) in different hydrological scenarios of Lake Hongze and Lake Luoma using bio-incubation experiments coupled with excitation-emission matrices and parallel factor analysis (EEMs-PARAFAC). The results showed that (1) Three fluorescent components were obtained using PARAFAC, including a humic-like C1, a tryptophan-like C2 and a tyrosine-like C3. (2) After 28 days of bio-incubation, the bioavailability of dissolved organic carbon (BDOC) in Lake Hongze and Lake Luoma in dry season (17%±4% and 15%±4%) was higher than that in flood season (5%±5% and 10%±7%), and the high %BDOC values of the two lakes were mainly distributed in the inflowing river mouths. (3) The specific absorbance at 254 nm SUVA254 and humic-like components in the two lakes during dry season was significantly higher than that pre-bio-incubation, and ΔSUVA254 and ΔC1 were negative. The tyrosine-like component was significantly lower than that pre-bio-incubation and ΔC3 was positive, indicating that the microorganisms preferentially utilized the less stable tyrosine-like component in the dry season, and produced more stable humic-like C1 and also increased the humification of the samples collected from the two lakes. (4) There were significant negative correlations between BDOC, %BDOC and ΔSUVA254, ΔC3, %ΔC3, respectively. These results indicated that CDOM optical composition directly affects its bioavailability in the two lakes.