Effective pretreatment process to improve enzymatic saccharification and decrease inhibitors generation is a key operation involved in the lignocellulosic bioconversion. The pretreatment of steam explosion associated with ammonium sulfite (SEAS) process was carried out to investigate the effect on enzymatic hydrolysis and fermentation production as a combinatorial pretreatment. Results showed that after pretreatment (1.0 MPa, 30 min, 20%w/w ammonium sulfite added), the phenolic inhibitors derived from lignin significantly removed (37.8%), which transformed to chemical humic acid (humic acid and fulvic acid) mostly. Sugar conversion (glucan (77.8%) and xylan (73.3%)) and ethanol concentration (40.8 g/L) of combinatorial pretreated samples were increased by 24.7% and 33.8%, respectively, compared with steam explosion (SE) pretreated samples. FT-IR and elemental analysis results indicated that the lignin structure changed and aromatization degree increased after SEAS pretreatment. In addition, the ratio of C/N decreased and compost maturity degree increased with the holding time. The effect on the growth of wheat seedlings of soluble fulvic acid solution from combinatorial pretreatment was investigated, where below 1% (w/w) concentration did contribute to growth. Therefore, one-step chemical pretreatment process could be provided for inhibitors removal, enzymatic saccharification increase, and chemical humic acid formation as well.