Although hemoadsorption combined with hemodialysis (HAHD) is increasingly used in patients undergoing maintenance hemodialysis (MHD), the optimal treatment duration remains uncertain. This study compared the efficacy and safety of 2-hour versus 4-hour HAHD sessions, supported by in vitro kinetic modeling. An in vitro recirculation system was used to assess the adsorption kinetics of parathyroid hormone (PTH), β2‑microglobulin (β2‑MG), and tumor necrosis factor‑alpha (TNF‑α) over 4 h. Subsequently, 92 stable MHD patients were prospectively enrolled and assigned to receive a single HAHD session of either 2 h (n = 45) or 4 h (n = 47). Pre- and post-treatment blood samples were analyzed to determine the reduction ratios of inflammatory cytokines (IL-8 and TNF-α) and middle molecular toxins (PTH and β2-MG). In vitro results confirmed time-dependent adsorption, with 4-hour removal ratios for PTH (93.27%), β2-MG (93.36%), and TNF-α (50.54%) being significantly higher than those at 2 h (84.36%, 82.00%, and 27.56%, respectively; all p < 0.05). Clinically, the 4-hour group achieved significantly higher median reduction ratios for the inflammatory cytokines IL-8 (81.64% vs. 69.51%) and TNF-α (43.94% vs. 22.33%) and for the middle molecular toxins PTH (59.67% vs. 55.12%) and β2-MG (21.79% vs. 14.20%). Both durations were well tolerated, with no significant safety concerns. These findings indicate that prolonging HAHD to 4 h improves the clearance of inflammatory and middle molecular weight toxins without compromising safety and may offer clinical benefits for MHD patients with high inflammatory burdens.