The fluorescence EEM technique, PARAFAC modeling, and hydrocarbon composition were used to characterize oil components and to examine the chemical evolution and degradation pathways of Macondo crude oil under controlled laboratory conditions. Three major fluorescent oil components were identified, with Ex/Em maxima at 226/328, 262/315, and 244/366 nm, respectively. An average degradation half-life of ∼20 d was determined for the oil components based on fluorescence EEM and hydrocarbon composition measurements, showing a dynamic chemical evolution and transformation of the oil during degradation. Dispersants appeared to change the chemical characteristics of oil, to shift the fluorescence EEM spectra, and to enhance the degradation of low-molecular-weight hydrocarbons. Photochemical degradation played a dominant role in the transformation of oil components, likely an effective degradation pathway of oil in the water column. Results from laboratory experiments should facilitate the interpretation of field-data and provide insights for understanding the fate and transport of oil components in the Gulf of Mexico.