This study investigated the recycle utilization of SPORL pretreatment spent liquor. Three lignosulfonates (LSs) were purified from the spent liquor of SPORL pretreated Beetle-killed lodgepole pine (BKLP), Poplar NE222 (NE222), and Douglas-fir (FS10). The structural characterization showed that the apparent molecular mass and sulfur content of NE222-LS were lowest, but the phenolic group content was highest. FS10-LS, from a pH profiling SPORL pretreatment, had the highest apparent molecular mass but medium sulfur and phenolic group content. The spectral analyses exhibited that the guaiacyl unit was the main structure in BKLP and FS10 LSs, while NE222-LS mainly contained both guaiacyl and syringyl units. Both LSs and SPORL pretreatment spent liquors were used as additives to enzymatic hydrolysis of Whatman paper and ethanol production from glucose. LSs and liquors, from SPORL pretreated BKLP and NE222, could obviously enhance the enzymatic saccharification. Nevertheless, LS and liquor from SPORL pretreated FS10 presented a slight negative effect on enzymatic saccharification. All LSs and liquors with low concentration exhibited no inhibition on ethanol fermentation from glucose. When whole spent liquors without any detoxification were applied to prepare the fermentation medium with an initial glucose concentration of 100 g/L, the ethanol yield was almost the same as the control for BKLP and FS10 liquors. Nevertheless, the whole NE222 liquor without detoxification inhibited ethanol production thoroughly.