A red pine fast pyrolysis bio-oil was subjected to sequential solvent fractionation into n-hexane soluble (HS), ether soluble (ES), ether insoluble (EIS), dichloromethane soluble (DS), and methanol soluble (MeS) fractions. The volatile components of bio-oil were analyzed by gas chromatography-mass spectrometry (GC-MS), indicating the presence of acids, aldehydes, ketones, alcohols, phenols, and anhydromonosaccharides, which consisted of methoxy, hydroxy, and carbonyl functional groups. These results imply that the bio-oil was similar to the most reported fast pyrolysis bio-oil samples in molecular composition. The bio-oil and its five subtractions were analyzed by negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The predominant compounds in bio-oil were O-2-O-17 class species with 1-22 double-bond equivalent (DBE) values and 4-39 carbon numbers. The most abundant class species in biocrude oil, HS, ES, EIS, DS, and MeS subfractions were O-7, O-6, O-8, O-10, O-7, and O-8 class species, respectively. The predominant EIS subfraction presented an obvious relative low DBE value, sustaining the tentative identification as "sugar fraction". The predominant compounds in DS subfraction were likely lignin dimers, whereas those in MeS subfraction should be lignin dimers and trimers. The number of oxygen atoms of the bio-oil compounds was negatively correlated with the average DBE value, indicating that oxygen atoms were present in various functional groups of the bio-oil compounds. The NIOx class species were also identified, which contained 1-16 DBE and 6-30 carbon numbers.