This study evaluated the effects of chemical and organic fertilization on aboveground biomass and soil properties across three natural grassland regions in the middle section of the Greater Khingan Mountains. Field experiments were conducted in Keyouqian Banner, Keyouzhong Banner, and Zhalaite Banner using three treatments: fencing enclosure (control), fencing enclosure + chemical fertilizer, and fencing enclosure + organic fertilizer, each with three biological replicates. Both chemical and organic fertilization significantly increased aboveground biomass by 30.41% and 24.56%, respectively (p < 0.05), with no significant difference between treatments. Chemical fertilization significantly reduced soil available phosphorus by 9.01% (p < 0.05), whereas organic fertilization had no significant effect on the measured soil properties (p > 0.05). Aboveground biomass and soil properties differed among the three regions, and these differences were associated with variations in soil pH and background soil conditions. These findings indicate that fertilization effectively enhanced grassland productivity, whereas biomass and soil responses varied among regions with different soil conditions, highlighting the importance of considering regional soil characteristics when interpreting fertilization responses and developing grassland management strategies.