A continuous screw extrusion steam explosion (SESE) pretreatment method was used to disrupt the structure of eucalyptus fiber. The effects of increasing numbers of SESE pretreatment cycles on the structure and composition of eucalyptus fiber were investigated. Lignin was isolated from the eucalyptus fiber via the phosphoric acid method under modest reaction conditions (50 °C), and was characterized. The SESE pretreatment led to a decrease in crystallinity and degraded the hemicellulose and cellulose. The surface of the eucalyptus fiber was damaged by SESE pretreatment, and significant surface debris were observed. The isolated lignin was typical of guaiacyl-syringyl (G-S)-enriched lignin of eucalyptus hardwood. The SESE pretreatment led to the cleavage of ether linkages and condensation of lignin, and thus a more heterogeneous structure compared with that of raw lignin. The increased molecular weight of SESE-pretreated lignin showed that condensation was more pronounced with increased numbers of SESE cycles. The thermostability of lignin increased after three SESE cycles and remained stable with further cycles. This corresponded to the trend in the molecular weight of lignin with increased SESE pretreatment. The SESE pretreatment was more efficient for pretreating eucalyptus fiber than conventional batch-type steam explosion, and thus is more suitable for industrial application.