Dissolved Organic Matter (DOM) in biochars is important to carbon dynamics and contaminant transport in soils. Fluorescence excitation-emission matrices (EEMs) have been widely used to characterize dissolved organic matter (DOM). In this study, fluorescence regional integration (FRI) and parallel factor analysis (PARAFAC) applied to EEM allows good quantitative assessment of the composition of DOM derived from manure biochars. Manure biochars were produced using four types of manure, chicken, pig, cow, and sheep manure under various pyrolysis temperatures (300–600 °C) and holding times (0–120 min). The results from the determination of dissolved organic carbon (DOC), SUVA₂₅₄, and humification index (HIX) reflected that high pyrolysis temperature and long holding time led to a significant decrease in DOM quantity, aromaticity and humification. The FRI result showed that the pyrolysis process of DOM released from manure biochars included three changes, aromatic protein-like substance and microbial by-product-like substance generation (300–600 °C), fulvic-acid like substance decomposition (300–500 °C) and humic acid-like substance decomposition (600 °C). The PARAFAC modeling result showed that the pyrolysis process of DOM released from manure biochars contained two changes: three high molecular-weight humic-like substances decomposition and a low molecular-weight humic-like substance generation. The effect of the holding time on biochar-DOM is more significant at higher pyrolysis temperatures than lower pyrolysis temperatures. The correlation analysis result revealed that the generation of aromatic proteins, microbial by-products and fulvic acid came from the decomposition of humic-like substances including marine humic-like, UVA humic-like, and UVC + UVA humic-like substances. The results obtained in this study would be beneficial to guide the rational production and application of manure biochars.