A combination of fluorescence excitation-emission (EEM) spectrophotometry with parallel factor analysis (PARAFAC) and UV–vis spectroscopy was used to assess dissolved organic matter (DOM) release by the wheat straw-derived biochar in various environmental conditions. The results showed that the concentration of dissolved organic carbon (DOC) extracted from the biochar ranged from 435 to 5000mgkg−1, and the high DOM release would occur under the high temperature and alkaline environmental conditions. Four humic acid-like substances (C1, C2, C4 and C5), one fulvic acid-like substance (C3) and one tryptophan-like substance (C6) were identified by PARAFAC model. The distribution of the PARAFAC components in DOM was distinctively different among various extracts, and mainly composed of humic acid-like and fulvic acid-like material (accounting 55–78% and 10–37%). The high temperature enhanced the high release of humic acid-like DOM from the biochar. Humification index (HIX) of DOM released from the biochar in various extracts at the temperature of 20°C (0.99–1.09) was lower than that at the temperature of 60°C (1.31–1.92). The results obtained in the present study would be beneficial to understand biochar’s effects and processes in the possible environmental applications.