Fibrin glue-soaked gelatin sponge (FGGS) has been used for tissue sealing in neurosurgical practice, but too rapid clotting of fibrin glue occasionally prevents good fixation of FGGS. Dilution of thrombin may provide adequate manipulation time between mixing fibrinogen and thrombin on gelatin sponge and application into the tissue defects. The present study characterized the effect of thrombin dilution on the adhesion strength of FGGS and retrospectively assessed the clinical usage of the dilution for filling dead space or sealing arachnoid defect in 255 cases who underwent transsphenoidal surgery for the last 66 months. FGGS was prepared using three different concentrations of thrombin: 250 (standard), 50 (1:5 dilution), and 25 (1:10 dilution) units/ml, and incubated for three different periods (5, 20, and 60 seconds). FGGSs were applied over two adjacently positioned porcine skins placed on two metallic plates. The adhesion strength was evaluated by measuring maximum tensile strength during pulling out the sliding plate at a constant rate of displacement. The maximum adhesion strength was greater for FGGS with 1:10 diluted thrombin solution than for FGGS prepared with higher concentrations (p < 0.05). Adhesion strength did not decay for 20 seconds after the mixture. Only four of 255 cases (1.6%) required second reconstruction of sella floor due to the cerebrospinal fluid leakage. FGGS prepared with diluted thrombin solution can provide adequate adhesion strength for clinical use.