The bioavailability of dissolved organic carbon (DOC), i.e., %BDOC, has received increasing attention in recent years. The %BDOC level directly affects the biogeochemical behavior of organic matter as well as the toxicity and bioavailability of heavy metals and pollutants. Lake Taihu Basin, situated in the economically advanced Yangtze River Delta region of China, has faced severe pollution due to large-scale industrial and domestic wastewater discharge over recent decades, posing a significant threat to drinking water safety for residents. Investigating the %BDOC in Lake Taihu is crucial for understanding lake carbon cycling and ensuring safe water supplies in the region. Long-term bio-incubation experiments were conducted to explore the spatial and temporal variations and the main factors influencing %BDOC in Lake Taihu from August 2018 to May 2021. The study revealed that %BDOC in Lake Taihu was higher in February and May compared to August and November, following a decreasing trend from northwest to southeast, with the highest levels observed in Zhushan Bay. After 28 days of bioincubation, tryptophan-like C2 and red-shifted tyrosine-like C3 levels showed significant reductions compared to pre-incubation levels (t-test, p<0.001). The fluorescence intensity of protein-like components was found to directly influence %BDOC in Lake Taihu, and these components exhibited high bioavailability. Pearson correlation analysis, random forest modeling, and partial least squares (PLS) regression were employed to comprehensive analyze %BDOC and its associated physicochemical parameters. Results indicated that %BDOC was significantly and negatively correlated with SUVA254 (aromaticity level) and positively correlated with DOC, COD, TN, and TP. Dissolved oxygen was identified as a key indicator of %BDOC variations in Lake Taihu. Enhanced research and monitoring of lake %BDOC are pivotal for elucidating organic matter dynamics, assessing carbon emission potential, and protecting water supplies.